论文部分内容阅读
Abstract In this study， chalcone synthase and chalcone isomerase encoding genes （GhCHS and GhCHI） were cloned from upland cotton （Gossypium hirsutum） cultivar HM-40， and analyzed bioinformatically. Their functions were analyzed through virus induced gene silencing （VIGS）. The results showed that GhCHS gene has an open reading frame （ORF） of 1 170 bp and encodes a protein of 389 amino acids. Many phosphorylation sites were detected in GhCHS protein， suggesting that it may be involved in kinase phosphorylation. The deduced GhCHS protein was most closely related to Theobroma cacao CHS protein according to phylogenetic analysis. The GhCHI ORF was 630 bp and encoded a protein of 209 amino acids. Many phosphorylation sites were found in GhCHI protein， indicating that it may be related to kinase phosphorylation. The GhCHI protein was most closely related to Hibiscus cannabinus CHI protein according to phylogenetic analysis. Quantitative PCR （qPCR） showed that GhCHS and GhCHI were rapidly activated after inoculation with Verticillium dahliae VD07， and then their expression levels kept increasing over time， indicating that the two genes might play an important role in the defense response against Verticillium wilt. Virus induced gene silencing （VIGS） was used to silence endogenous GhCHS and GhCHI genes in upland cotton plants before VD07 was inoculated to identify disease resistance. The results showed that the disease index of plants untreated with Agrobacterium tumefaciens was 31.2， and that of the plants inoculated with empty vector was 30.0. The disease index of GhCHS-silenced plants was 72.5， and that in GhCHI-silenced plants was 67.5. These results confirmed that GhCHS and GhCHI may play an important role in defense response of upland cotton to Verticillium wilt.
Key words Upland cotton （Gossypium hirsutum L.）； Grafting； GhCHS； GhCHI； Verticillium wilt； VIGS
As an important cash crop， the production of cotton is related to the economic development of China[1]. However， Verticillium wilt， a widespread disease caused by Verticillium spp.， has caused serious losses to cotton production[2]. In order to improve cotton quality and yield， an increasing effort has been made to improve the resistance of cotton to Verticillium wilt.
Chalcone synthase （CHS） and chalcone isomerase （CHI） are the key enzymes in the synthesis of flavonoids in plant secondary metabolism[3-4]， and also play important roles in the phenylalanine metabolism and disease resistance of plants[5-6]. With strong adaptability and high resistance， many plant species are able to respond actively to multiple threats， by regulating the expression of corresponding genes. The study of Cui et al.[7] showed that CHS gene was greatly up-regulated in sorghum seedlings exposed to Bipolaris maydis， and its expression level in low resistant varieties was much lower than that in highly resistant varieties[7]. Chalcone isomerase has been found to play a role in regulating the metabolism of flavonoids[8]. Previous studies on Verticillium wilt resistant cotton were about the physiological and biochemical characteristics， cloning of disease-resistant genes， identification of disease-resistant proteins and epigenetics. Identifying the genes activated by the invasion of Verticillium dahliae may able to reveal the signaling pathway that leads to the defense responses of cotton plants against Verticillium wilt[9]. In this study， the full-length open reading frames （ORFs） of GhCHS and GhCHI genes were cloned from G. hirsutum and analyzed bioinformatically. Their expression levels in grafted cotton seedlings different days after the inoculation with V. dahliae were determined by qPCR. Moreover， the disease index of grafted cotton seedlings was investigated by individually silencing GhCHS and GhCHI genes using virus induced gene silencing （VIGS）， to provide a theoretical basis for studying the functions of the two genes[10].
Materials and Methods
Materials
A susceptible cultivar HM-40 of upland cotton， and a highly resistant cultivar 7124 of sea island cotton， provided by the laboratory of College of Life Sciences， Wuhan University， were selected as the experimental material in this study. The seedlings of HM-40 were grafted onto the rootstock of 7124 after their second true leaf expanded. Seven days after grafting， V. dahliae VD07， a highly virulent pathogen， which was kindly provided by Heqin ZHU from the State Key Laboratory of Cotton Biology， Institute of Cotton Research， Chinese Academy of Agricultural Sciences， was inoculated into the graftings survived， at a dosage of 20 ml （107 pfu/ml） per seedling. Then， the true leaves of the graftings were sampled 0， 2， 4， 6， 8 and10 d after the inoculation， and stored at -80 ℃ till analysis.
According to Gao et al.[11]， Agrobacterium-mediated VIGS assay was performed 7 d after grafting to analyze gene functions. In detail， 16 d after Agrobacterium tumefaciens strains transformed with pYL-156-pYL-192， pYL-156-GhCHS-pYL-192 or pYL-156-GhCHI-pYL-192 were inoculated， the true leaves of the grafted cotton seedlings were collected and stored at -80 ℃ for later tests. Meanwhile， V. dahliae VD07 was inoculated at a dosage of 20 ml （107 pfu/ml） per seedling to the seedlings that had been inoculated with A. tumefaciens. And 25 d the inoculation of V. dahliae VD07， disease index of the seedlings in each treatment was measured.
RNAprep Pure Plant Kit DP441 was purchased from Tiangen Biotech （Beijing） Co.， Ltd. PrimeScriptTM RT reagent Kit with gDNA Eraser and pMD18-T vector were purchased from TaKaRa Biotechnology （Dalian） Co.， Ltd. OMEGA E.Z.N.A. Cycle Pure Kit， EsTaq mix， competent E. coli Top10 were purchased from Wuhan Ouruiwen Biotech Co.， Ltd. Kod plus neo was purchased from TOYOBO Biotechnology （Shanghai） Co.， Ltd. Competent A. tumefaciens GV3101 was prepared in our laboratory. Tobacco rattle virus （TRV）-derived VIGS vectors pYL-192 and pYL-156 were kindly provided by Professor Yule LIU from Tsinghua University. Total RNA extraction and cDNA preparation
Total RNA was extracted with RNAprep Pure Plant Kit DP441， and reverse-transcribed into the first-strand cDNA using TAKARA PrimeScriptTM RT Reagent Kit with gDNA Eraser.
Cloning of GhCHS and GhCHI genes
Primers were designed using Oligo 7. GhCHS gene was cloned with the Kod-Plus-Neo kit and the primers GhCHS-F （5′-AGCATAGCAGCTTAGTCCA-3′） and GhCHS-R （5′-AACTGAATAGGCGTTAAGCTTC-3′）. The PCR was started with pre-denaturation at 94 ℃ for 5 min， followed by 28 cycles of denaturation at 98 ℃ for 15 s， annealing at 55 ℃ for 30 s， and extension at 68 ℃ for 1 min； the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min. GhCHI gene was amplified with the primers GhCHI-F （5′-CTTGCCTGCCTTAAACCTC3′） and GhCHI-R （5′-TCATTTTGGGCTTCACTCA-3′） by the same way described above. The PCR products were run on a 1% agarose gel and purified with Omega Gel Extraction Kit following the manufacture′s instruction. The purified DNA fragments were ligated into pMD18-T vector， and transformed into competent E. coli Top10 cells， and cultured overnight. Single colonies were subjected to colony PCR and sequenced by Suzhou GENEWIZ Biotechnology Co.， Ltd.
Construction VIGS vectors of GhCHS and GhCHI genes
GhCHS gene was amplified with VIGS primers pYL-156-CHS-F （5′-CGCGGATCCAGCATAGCAGCTTAGTCCA-3′） and pYL-156-CHS-R （5′-CCGCTCGAGAACTGAATAGGCGTTAAGC-TTC-3′） （the introduced restriction sites were underlined） in the presence of LA Taq DNA polymerase. The PCR program was pre-denaturation at 94 ℃ for 5 min， followed by 30 cycles of denaturation at 94 ℃ for 30 s， annealing at 55 ℃ for 30 s， and extension at 72 ℃ for 1.5 min， and completed by a final extension step at 72 ℃ for 10 min.
GhCHI gene was amplified by the same way using the VIGS primers pYL-156-CHI-F （5′-CGCGGATCCCTTGCCTGCCTTAAACCTC-3′） and pYL-156-CHI-R （5′-CCGCTCGAGTCATTTTGGGCTTCACTCA-3′） （the introduced restriction sites were underlined）. The amplification products were run on a 1% agarose gel， and purified with Omega Gel Extraction Kit following the manufacture′s instruction. The purified DNA fragments and the plasmid pYL-156 were separately digested. The ligation reaction was carried out with solution I for 4 h. The ligation products were transformed into competent E. coli Top10 cells， and cultured overnight. Single colonies were subjected to colony PCR and sequenced by Suzhou GENEWIZ Biotechnology Co.， Ltd.
Positive plasmids were extracted， electrotransformed into competent A. tumefaciens GV3101 cells， inoculated into plates， and cultured overnight at 28 ℃ in the dark. Colony PCR was carried out to screen pYL-156-GhCHS and pYL-156-GhCHI positive colonies. VIGS assay was conducted as previously described[11]. Bioinformatics analysis of cloned gene sequences
The physicochemical properties of the two proteins were deduced using Protparam （http：//web.expasy.org/protparam/） and their subcellular localization was predicted using PSORT （http：//www.genscript.com/psort.html）. SignalP 4.1 was used predict the presence of the signal peptide， TMHMM to predict transmembrane helices. Then the amino acid sequence was searched with protein Blast at the NCBI. Phylogenetic tree was constructed based on GhCHS or GhCHI proteins using MEGA 5 software. The phosphorylation sites of the proteins were predicted using NetPhos 2.0 program.
Expression analysis of GhCHS and GhCHI genes
The relative expression levels of GhCHS and GhCHI genes were analyzed by qPCR according to the instructions of the PrimeScriptTM RT reagent Kit with gDNAEraser， using actin as the reference gene. The primers were qCHS-F （5′-TTCAACCATTGGGCATATCCG- 3′）， qCHS-R （5′- ACTCTGAAAGAACGTGCCTTG-3′）， qCHI-F （5′-CCATTTTCCAGCAAATTCAGC-3′） and qCHI-R （5′-TTTCTTGATCATCTCGACCAC-3′）， actin-F （5′-TCACGGAAGCACCTCTCAAC-3′） and actin-R （5′-ACAAAGAGAGAACGGCCTGG-3′）. Three technical replicates and three biological replicates were prepared for each sample. The qPCR reaction was run on a Bio-Rad CFX96 Manager， and the relative expression level of the genes was calculated with 2^-ΔΔCt method. The qPCR was started with pre-denaturation at 95 ℃ for 30 s， followed by 40 cycles of denaturation at 95 ℃ for 5 s， annealing and extension at 57 ℃ for 20 s. After that， the temperature was increased from 65 to 95 ℃ at a rate of 0.5 ℃ per cycle to yield the dissociation curves.
Quantitative or semi-quantitative PCR analysis of GhCHS and GhCHI genes in VIGS treated cotton seedlings
The cDNA stock of cotton was serially diluted by 10-fold， and semi-quantitatively analyzed with primers V-bCHS-F （5′-TTGGATGAGATGAGGAA-3′） and V-bCHS-R （5′-CAAAGTAACTTATTATTGCC-3′）， V-bCHI-F （5′-CTGTGAGGGACCGATTG-3′） and V-bCHI-R （5′-TGGCATTTTCATTTTGG-3′）， actinB-F （5′-GTTATGGTTGGGATGGG-3′） and actinB-R （5′-CAAGGTCAAGACGGAGG-3′）， following the instructions of the PrimeScriptTM RT reagent Kit with gDNA Eraser. The PCR program was pre-denaturation at 94 ℃ for 5 min， followed by 20， 25， 28， 31， 32， 33， 34， 35， 36， 37 or 38 cycles of denaturation at 94 ℃ for 30 s， annealing at 55 ℃ for 30 s， and extension at 72 ℃ for 20 s， and completed by a final extension step at 72 ℃ for 10 min. The products were electrophoresed on a 1% agarose gel. The relative expression levels of GhCHS and GhCHI genes were analyzed by PCR. The primers were V-qCHS-F （5′-TTTTACCATTCATAGCATAG-3′） and V-qCHS-R （5′-TACGGAAATAGTAGTCAGG-3′）， V-qCHI-F （5′-ACAAGGGGAGTGTCTGC-3′） and V-qCHI-R （5′-TGGCATTTTCATTTTGG-3′）， actin-F （5′-TCACGGAAGCACCTCTCAAC-3′） and actin-R （5′-ACAAAGAGAGAACGGCCTGG-3′）. The qPCR procedures were the same as described above.
Chengpan SONG et al. Cloning and Expression Analysis of Chalcone Synthase and Chalcone Isomerase Encoding Genes in Gossypium hirsutum
Results and Analysis
The GhCHS and GhCHI genes cloned from G. hirsutum
A DNA fragment of about 1 400 bp was PCR amplified using the reverse-transcribed cDNA of G. hirsutum as the template， in the presence of the primers GhCHS-F （5′-AGCATAGCAGCTTAGTCCA -3′） and GhCHS-R （5′-AACTGAATAGGCGTTAAGCTTC-3′） （Fig. 1）. Sequencing analysis showed that open reading frame （ORF） of the fragment was 1 170 bp （Fig .2）. Another DNA fragment of about 800 bp was PCR amplified by the same way， using the primers GhCHI-F （5′-CTTGCCTGCCTTAAACCTC-3′） and GhCHI-R （5′-TCATTTTGGGCTTCACTCA-3′）. Sequencing analysis showed that it had an ORF of 630 bp （Fig. 3）.
Physical and chemical properties of GhCHS and GhCHI proteins
Physical and chemical properties of GhCHS protein
The physical and chemical properties of GhCHS protein were analyzed using Protparam server. The results showed that GhCHS protein has a chemical formula of C1 900H3 045N507O563S19， a theoretical isoelectric point of about 6.12 and a molecular mass of about 42.608 kDa. As shown in Table 1， the protein is rich in Leu， Val， Ala， Gly， Glu and Lys. There are 48 negatively charged acidic amino acids （Glu and Asp） and 44 positively charged basic amino acids （Lys And Arg）， indicating that GhCHS protein is a negatively charged acidic protein. Its instability index is predicted to be 38.76. Its estimated half-life is about 30 h when expressed in vitro in mammalian reticulocytes， over 20 h when expressed in yeast cells， and 10 h when expressed in E. coli， indicating that it is a stable protein.
Physical and chemical properties of GhCHI protein
The physical and chemical properties of GhCHI protein were analyzed using Protparam server. The results showed that GhCHI protein has a chemical formula of C1 059H1 662N262O322S5， a theoretical isoelectric point of about 4.86 and a molecular mass of about 23.377 kDa. As shown in Table 2， the protein is rich in Glu， Val， Lys， Ala， Leu and Thr. There are 34 negatively charged acidic amino acids （Glu and Asp） and 22 positively charged basic amino acids （Lys And Arg）， indicating that GhCHS protein is a negatively charged acidic protein. Its instability index is predicted to be 35.39. Its estimated half-life is about 30 h when expressed in vitro in mammalian reticulocytes， over 20 h when expressed in yeast cells， and 10 h when expressed in E. coli， indicating that it is a stable protein. Hydrophobicity and subcellular localization of GhCHS and GhCHI proteins
Hydrophobicity and subcellular localization of GhCHS protein
The hydrophobicity of GhCHS protein was predicted using ProtScale （http：//web.expasy.org/protscale/）. As shown in Fig. 4， GhCHS protein has a grand average of hydropathicity （GRAVY） of -0.096， indicating that the entire protein is hydrophilic. The subcellular localization of GhCHS protein was predicted using PSORT. The results showed that the GhCHS protein may be located in the cytoplasm （with a score of 0.450）， peroxisome （with a score of score 0.300）， mitochondrial matrix （with a score of 0.100） and lysosome （with a score of 0.100） in cells. The presence of signal peptide of GhCHS protein was analyzed using SignalP 4.1 software， and the result showed there is no signal peptide sequence in GhCHS protein， so that the protein cannot pass through the cell membrane. The presence of transmembrane domain of the protein was predicted using TMHMM， and the results proved that there is no transmembrane helix in the protein. In summary， GhCHS protein is inferred to be a hydrophilic cytoplasmic protein.
Hydrophobicity and subcellular localization of GhCHI protein
The hydrophobicity of GhCHI protein was predicted using ProtScale （http：//web.expasy.org/protscale/）. As shown in Fig. 5， GhCHI protein has a grand average of hydropathicity （GRAVY） of -0.153， indicating that the entire protein is hydrophilic. The subcellular localization of GhCHS protein was predicted using PSORT， which revealed GhCHS protein may be located in the cytoplasm （with a score of 0.450）， peroxisome （with a score of 0.206）， lysosome （with a score of 0.100） and mitochondrial matrix （with a score of 0.100）. The presence of signal peptide of GhCHI protein was analyzed using SignalP 4.1 software， and the result showed there was no signal peptide sequence in this protein， indicating that it cannot pass through the cell membrane. TMHMM predicted that the protein has no transmembrane helix. Based on above results， GhCHI protein is a hydrophilic cytoplasmic protein.
Sequence alignment and phylogenetic analysis of GhCHS and GhCHI proteins
Sequence alignment and phylogenetic analysis of GhCHS protein
BlastP search revealed that the amino acid sequence of GhCHS of G. hirsutum has high similarity with that of other plant species. Then， amino acid sequences of the protein of G. hirsutum， Theobroma cacao （XP_007034442.1）， Hibiscus cannabinus （AIC75908.1）， Actinidia chinensis （AGV53049.1）， Camellia japonica （BAI66465.1）， Prunus avium （AJO67963.1）， Arabidopsis thaliana （NP_196897.1）， Solanum tuberosum （AEN83501.1）， Oryza sativa Japonica Group， （NP_001068009.1）， Pyrus pyrifolia （AFQ92052）. 1）， Rhus chinensis （AGH13332.1）， Nelumbo nucifera （ADD74167.1） and Malus domestica （BAB92996.1） were aligned using MEGA 5. The results showed that there is a high similarity in amino acid sequence of GhCHS protein between G. hirsutum and other plant species （Fig. 6）. Phylogenetic analysis indicated that GhCHS protein has the closest relationship with that of T. cacao （XP_007034442.1） （Fig. 7）. Sequence alignment and phylogenetic analysis of GhCHI protein
BlastP search revealed that the amino acid sequence of GhCHI of G. hirsutum had high similarity with that of other species. Then， amino acid sequences of the protein of G. hirsutum， T. cacao （XP_007011312.1）， H. cannabinus （AIC73814.1）， A. chinensis （AGV53050.1）， P. pyrifolia （AFQ92051.1）， Actinidia chrysantha （AIY53017.1）， Phyllanthus emblica （AHA61355.1）， Garcinia mangostana （ACM62743.1）， Lonicera japonica （AGE10598.1）， Lycium ruthenicum （AHH86093.1）， Arachis hypogaea （AEO17326.1）， Indosasa hispida （AHC07954.1）， Lycium chinense （AIC33515.1） and Morus alba （ALD83622.1） were aligned using MEGA 5.0. The results revealed that there is a high similarity in amino acid sequence of GhCHI protein between G. hirsutum and other plant species （Fig. 8）. Phylogenetic analysis indicated that GhCHI protein has the closest relationship with that of H. cannabinus （AIC73814.1） （Fig. 9）.
Phosphorylation sites of GhCHS and GhCHI proteins
Phosphorylation sites of GhCHS protein
Protein phosphorylation， a post-translational modification of proteins， participates in and regulates many cell activities. Phosphorylation alters the conformation， stability， activity and location of a protein in the cell[12]. GhCHS protein contains 24 threonine （Thr） and 22 serine （Ser） residues， which are potential phosphorylation sites. NetPhos 2.0 predicted that there are 17 potential phosphorylation sites in GhCHS protein including six Thr phosphorylation sites， nine Ser phosphorylation sites and two tyrosine （Tyr） phosphorylation sites （Fig. 10）， suggesting that GhCHS protein may be involved in kinase phosphorylation.
Phosphorylation sites of GhCHI protein
GhCHI protein contains 15 Thr and 10 Ser residues. NetPhos 2.0 predicted that there are eight potential phosphorylation sites in GhCHI protein including three Thr phosphorylation sites， three Ser phosphorylation sites and two tyrosine （Tyr） phosphorylation sites （Fig. 11）， suggesting that GhCHI protein may be involved in kinase phosphorylation.
Relative expression of GhCHS and GhCHI genes in G. hirsutum infected with V. dahliae VD07
The relative expression levels of GhCHS and GhCHI genes in true leaves of grafted G. hirsutum plants （7124 as the rootstock and HM-40 as the scion） were analyzed by qPCR. The results showed that the expression of GhCHS and GhCHI genes increased gradually over time after inoculation with V. dahliae VD07. The expression level of GhCHS gene on Day 2， 4， 6， 8 and 10 after the inoculation with V. dahliae VD07 was significantly higher than that on Day 0 （P<0.05）. The expression level of GhCHS gene on Day 10 was up-regulated by about 39 times， compared with that on Day 0. The expression level of GhCHI gene on Day 2， 4， 6， 8 and 10 after the inoculation with V. dahliae VD07 was also significantly higher than that on Day 0 （P<0.05）. The expression level of GhCHI gene on Day 10 was up-regulated by about 14 times， compared with that on Day 0. The results suggested that both GhCHS and GhCHI genes may play an important role in defense response of grafted cotton against Verticillium wilt. Function of GhCHS and GhCHI genes analyzed by VIGS
Sixteen days after the inoculation with A. tumefaciens transformed with pYL-156-pYL-192， pYL-156-GhCHS-pYL-192 or pYL-156-GhCHI-pYL -192， the total RNA of G. hirsutum leaves was extracted， reverse transcribed and subjected to quantitative and semi-quantitative PCR assays （Fig. 13）. The results showed that the expression levels of GhCHS and GhCHI in G. hirsutum leaves inoculated with VIGS vectors were decreased by 84.74% and 87.86%， respectively， compared with that in the leaves inoculated with empty vector （Fig. 14）. Twenty-five days after inoculation， the disease index of the grafted plants untransformed with A. tumefaciens was 31.2， indicating the plants were disease resistant； the disease index of the grafted plants transformed with empty vector was 30.0， indicating that the plants were disease resistant. The disease index of the grafted plants transformed with GhCHS VIGS vector was 72.5， and that in of the plants transformed with GhCHI VIGS vector was 67.5， indicating that the plants were disease susceptible （Fig. 15）.
Discussion
With development of plant genetic engineering， new technologies and bioinformatics， many genes of plants have been cloned， and their functions can be rapidly predicted and analyzed. VIGS is a fast and powerful method to study gene function in plants by silencing the gene of interest. Chalcone synthase and chalcone isomerase are the key enzymes in the synthesis of flavonoids， and involved in a variety of plant growth， stress and defense responses of plants. But their function in the resistance of cotton to Verticillium wilt has been rarely reported.
In this study， GhCHS and GhCHI genes were cloned from G. hirsutum， and analyzed bioinformatically. The results showed that both GhCHS and GhCHI proteins are hydrophilic cytoplasmic proteins， contains many phosphorylation sites， suggesting that they may be involved in kinase phosphorylation. In addition， both GhCHS and GhCHI genes are activated by the inoculation of V. dahliae， suggesting that they may be related to plant defense response. Using VIGS technology， we found that the silencing of either GhCHS or GhCHI gene lead to the loss of resistance to Verticillium wilt in cotton， which confirmed the function of the two genes in defense response against Verticillium wilt.
References
[1] WAKELYN PJ， N BERONIERE NR， FRENCH AD， et al. Cotton fiber chemistry and technology （International Fiber Science and Technology）[M]. USA： CRC Press， 2007. [2] BELL A A， HILLOCKS R J. Verticillium wilt.[M]. London： CAB International， 1992.
[3] ABDEL-LATEIF K， VAISSAYRE V， GHERBI H， et al. Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation[J]. New Phytologist， 2013， 199（4）： 1012-1021.
[4] MUIR SR， COLLINS GJ， ROBINSON S， et al. Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols[J]. Nature Biotechnology， 2001， 19， 470-474.
[5] BESSEAU S， LAURENT H， PIERRETTE G， et al. Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth[J]. The Plant Cell， 2007， 19： 148-162.
[6] KOES RE， FRANCESCA Q， JOSEPH NM. The flavonoid biosynthetic pathway in plants： Function and evolution[J]. Bioessays， 1994， 16： 123-132.
[7] CUI Y， FREDERIKSEN R， MAGILL C. Chalcone synthase and phenylalanine ammonialyase mRNA levels following exposure of sorghum seedlings to three fungal pathogens[J]. Physiological and Molecular Plant Pathology， 1996， 49（3）： 187-199.
[8] KAWASAKI T， KOEDUKA T， SUGIYAMA A， et al. Metabolic engineering of flavonoids with prenyltransferase and chalcone isomerase genes in tomato fruits[J]. Plant Biotechnology， 2014， 31（5）： 567-571.
[9] XU L， ZHU LF， TU LL， et al. Differential gene expressionin cotton defense response to Verticillium dahliae by SSH[J]. Journal of Phytopathology， 2011， 159（9）： 606-615.
[10] ROBERTSON D. Vigs vectors for gene silencing： Many targets， many tools[J]. Plant Biology， 2004， 55： 495-519.
[11] GAO XQ， JR RC B， SHAN LB， et al. Agrobacterium-mediated virus-induced gene silencing assay in cotton[J]. Journal of Visualized Experiments， 2011， 54： e2938.
[12] FANG ZZ， ZHOU DR， JIANG CC， et al. Cloning and bioinformatics analysis of Ran genes from Prunus salicina Lindl.[J]. Molecular Plant Breeding， 2014， 12（4）： 780-787.
Key words Upland cotton （Gossypium hirsutum L.）； Grafting； GhCHS； GhCHI； Verticillium wilt； VIGS
As an important cash crop， the production of cotton is related to the economic development of China[1]. However， Verticillium wilt， a widespread disease caused by Verticillium spp.， has caused serious losses to cotton production[2]. In order to improve cotton quality and yield， an increasing effort has been made to improve the resistance of cotton to Verticillium wilt.
Chalcone synthase （CHS） and chalcone isomerase （CHI） are the key enzymes in the synthesis of flavonoids in plant secondary metabolism[3-4]， and also play important roles in the phenylalanine metabolism and disease resistance of plants[5-6]. With strong adaptability and high resistance， many plant species are able to respond actively to multiple threats， by regulating the expression of corresponding genes. The study of Cui et al.[7] showed that CHS gene was greatly up-regulated in sorghum seedlings exposed to Bipolaris maydis， and its expression level in low resistant varieties was much lower than that in highly resistant varieties[7]. Chalcone isomerase has been found to play a role in regulating the metabolism of flavonoids[8]. Previous studies on Verticillium wilt resistant cotton were about the physiological and biochemical characteristics， cloning of disease-resistant genes， identification of disease-resistant proteins and epigenetics. Identifying the genes activated by the invasion of Verticillium dahliae may able to reveal the signaling pathway that leads to the defense responses of cotton plants against Verticillium wilt[9]. In this study， the full-length open reading frames （ORFs） of GhCHS and GhCHI genes were cloned from G. hirsutum and analyzed bioinformatically. Their expression levels in grafted cotton seedlings different days after the inoculation with V. dahliae were determined by qPCR. Moreover， the disease index of grafted cotton seedlings was investigated by individually silencing GhCHS and GhCHI genes using virus induced gene silencing （VIGS）， to provide a theoretical basis for studying the functions of the two genes[10].
Materials and Methods
Materials
A susceptible cultivar HM-40 of upland cotton， and a highly resistant cultivar 7124 of sea island cotton， provided by the laboratory of College of Life Sciences， Wuhan University， were selected as the experimental material in this study. The seedlings of HM-40 were grafted onto the rootstock of 7124 after their second true leaf expanded. Seven days after grafting， V. dahliae VD07， a highly virulent pathogen， which was kindly provided by Heqin ZHU from the State Key Laboratory of Cotton Biology， Institute of Cotton Research， Chinese Academy of Agricultural Sciences， was inoculated into the graftings survived， at a dosage of 20 ml （107 pfu/ml） per seedling. Then， the true leaves of the graftings were sampled 0， 2， 4， 6， 8 and10 d after the inoculation， and stored at -80 ℃ till analysis.
According to Gao et al.[11]， Agrobacterium-mediated VIGS assay was performed 7 d after grafting to analyze gene functions. In detail， 16 d after Agrobacterium tumefaciens strains transformed with pYL-156-pYL-192， pYL-156-GhCHS-pYL-192 or pYL-156-GhCHI-pYL-192 were inoculated， the true leaves of the grafted cotton seedlings were collected and stored at -80 ℃ for later tests. Meanwhile， V. dahliae VD07 was inoculated at a dosage of 20 ml （107 pfu/ml） per seedling to the seedlings that had been inoculated with A. tumefaciens. And 25 d the inoculation of V. dahliae VD07， disease index of the seedlings in each treatment was measured.
RNAprep Pure Plant Kit DP441 was purchased from Tiangen Biotech （Beijing） Co.， Ltd. PrimeScriptTM RT reagent Kit with gDNA Eraser and pMD18-T vector were purchased from TaKaRa Biotechnology （Dalian） Co.， Ltd. OMEGA E.Z.N.A. Cycle Pure Kit， EsTaq mix， competent E. coli Top10 were purchased from Wuhan Ouruiwen Biotech Co.， Ltd. Kod plus neo was purchased from TOYOBO Biotechnology （Shanghai） Co.， Ltd. Competent A. tumefaciens GV3101 was prepared in our laboratory. Tobacco rattle virus （TRV）-derived VIGS vectors pYL-192 and pYL-156 were kindly provided by Professor Yule LIU from Tsinghua University. Total RNA extraction and cDNA preparation
Total RNA was extracted with RNAprep Pure Plant Kit DP441， and reverse-transcribed into the first-strand cDNA using TAKARA PrimeScriptTM RT Reagent Kit with gDNA Eraser.
Cloning of GhCHS and GhCHI genes
Primers were designed using Oligo 7. GhCHS gene was cloned with the Kod-Plus-Neo kit and the primers GhCHS-F （5′-AGCATAGCAGCTTAGTCCA-3′） and GhCHS-R （5′-AACTGAATAGGCGTTAAGCTTC-3′）. The PCR was started with pre-denaturation at 94 ℃ for 5 min， followed by 28 cycles of denaturation at 98 ℃ for 15 s， annealing at 55 ℃ for 30 s， and extension at 68 ℃ for 1 min； the amplification was completed by holding the reaction mixture at 72 ℃ for 10 min. GhCHI gene was amplified with the primers GhCHI-F （5′-CTTGCCTGCCTTAAACCTC3′） and GhCHI-R （5′-TCATTTTGGGCTTCACTCA-3′） by the same way described above. The PCR products were run on a 1% agarose gel and purified with Omega Gel Extraction Kit following the manufacture′s instruction. The purified DNA fragments were ligated into pMD18-T vector， and transformed into competent E. coli Top10 cells， and cultured overnight. Single colonies were subjected to colony PCR and sequenced by Suzhou GENEWIZ Biotechnology Co.， Ltd.
Construction VIGS vectors of GhCHS and GhCHI genes
GhCHS gene was amplified with VIGS primers pYL-156-CHS-F （5′-CGCGGATCCAGCATAGCAGCTTAGTCCA-3′） and pYL-156-CHS-R （5′-CCGCTCGAGAACTGAATAGGCGTTAAGC-TTC-3′） （the introduced restriction sites were underlined） in the presence of LA Taq DNA polymerase. The PCR program was pre-denaturation at 94 ℃ for 5 min， followed by 30 cycles of denaturation at 94 ℃ for 30 s， annealing at 55 ℃ for 30 s， and extension at 72 ℃ for 1.5 min， and completed by a final extension step at 72 ℃ for 10 min.
GhCHI gene was amplified by the same way using the VIGS primers pYL-156-CHI-F （5′-CGCGGATCCCTTGCCTGCCTTAAACCTC-3′） and pYL-156-CHI-R （5′-CCGCTCGAGTCATTTTGGGCTTCACTCA-3′） （the introduced restriction sites were underlined）. The amplification products were run on a 1% agarose gel， and purified with Omega Gel Extraction Kit following the manufacture′s instruction. The purified DNA fragments and the plasmid pYL-156 were separately digested. The ligation reaction was carried out with solution I for 4 h. The ligation products were transformed into competent E. coli Top10 cells， and cultured overnight. Single colonies were subjected to colony PCR and sequenced by Suzhou GENEWIZ Biotechnology Co.， Ltd.
Positive plasmids were extracted， electrotransformed into competent A. tumefaciens GV3101 cells， inoculated into plates， and cultured overnight at 28 ℃ in the dark. Colony PCR was carried out to screen pYL-156-GhCHS and pYL-156-GhCHI positive colonies. VIGS assay was conducted as previously described[11]. Bioinformatics analysis of cloned gene sequences
The physicochemical properties of the two proteins were deduced using Protparam （http：//web.expasy.org/protparam/） and their subcellular localization was predicted using PSORT （http：//www.genscript.com/psort.html）. SignalP 4.1 was used predict the presence of the signal peptide， TMHMM to predict transmembrane helices. Then the amino acid sequence was searched with protein Blast at the NCBI. Phylogenetic tree was constructed based on GhCHS or GhCHI proteins using MEGA 5 software. The phosphorylation sites of the proteins were predicted using NetPhos 2.0 program.
Expression analysis of GhCHS and GhCHI genes
The relative expression levels of GhCHS and GhCHI genes were analyzed by qPCR according to the instructions of the PrimeScriptTM RT reagent Kit with gDNAEraser， using actin as the reference gene. The primers were qCHS-F （5′-TTCAACCATTGGGCATATCCG- 3′）， qCHS-R （5′- ACTCTGAAAGAACGTGCCTTG-3′）， qCHI-F （5′-CCATTTTCCAGCAAATTCAGC-3′） and qCHI-R （5′-TTTCTTGATCATCTCGACCAC-3′）， actin-F （5′-TCACGGAAGCACCTCTCAAC-3′） and actin-R （5′-ACAAAGAGAGAACGGCCTGG-3′）. Three technical replicates and three biological replicates were prepared for each sample. The qPCR reaction was run on a Bio-Rad CFX96 Manager， and the relative expression level of the genes was calculated with 2^-ΔΔCt method. The qPCR was started with pre-denaturation at 95 ℃ for 30 s， followed by 40 cycles of denaturation at 95 ℃ for 5 s， annealing and extension at 57 ℃ for 20 s. After that， the temperature was increased from 65 to 95 ℃ at a rate of 0.5 ℃ per cycle to yield the dissociation curves.
Quantitative or semi-quantitative PCR analysis of GhCHS and GhCHI genes in VIGS treated cotton seedlings
The cDNA stock of cotton was serially diluted by 10-fold， and semi-quantitatively analyzed with primers V-bCHS-F （5′-TTGGATGAGATGAGGAA-3′） and V-bCHS-R （5′-CAAAGTAACTTATTATTGCC-3′）， V-bCHI-F （5′-CTGTGAGGGACCGATTG-3′） and V-bCHI-R （5′-TGGCATTTTCATTTTGG-3′）， actinB-F （5′-GTTATGGTTGGGATGGG-3′） and actinB-R （5′-CAAGGTCAAGACGGAGG-3′）， following the instructions of the PrimeScriptTM RT reagent Kit with gDNA Eraser. The PCR program was pre-denaturation at 94 ℃ for 5 min， followed by 20， 25， 28， 31， 32， 33， 34， 35， 36， 37 or 38 cycles of denaturation at 94 ℃ for 30 s， annealing at 55 ℃ for 30 s， and extension at 72 ℃ for 20 s， and completed by a final extension step at 72 ℃ for 10 min. The products were electrophoresed on a 1% agarose gel. The relative expression levels of GhCHS and GhCHI genes were analyzed by PCR. The primers were V-qCHS-F （5′-TTTTACCATTCATAGCATAG-3′） and V-qCHS-R （5′-TACGGAAATAGTAGTCAGG-3′）， V-qCHI-F （5′-ACAAGGGGAGTGTCTGC-3′） and V-qCHI-R （5′-TGGCATTTTCATTTTGG-3′）， actin-F （5′-TCACGGAAGCACCTCTCAAC-3′） and actin-R （5′-ACAAAGAGAGAACGGCCTGG-3′）. The qPCR procedures were the same as described above.
Chengpan SONG et al. Cloning and Expression Analysis of Chalcone Synthase and Chalcone Isomerase Encoding Genes in Gossypium hirsutum
Results and Analysis
The GhCHS and GhCHI genes cloned from G. hirsutum
A DNA fragment of about 1 400 bp was PCR amplified using the reverse-transcribed cDNA of G. hirsutum as the template， in the presence of the primers GhCHS-F （5′-AGCATAGCAGCTTAGTCCA -3′） and GhCHS-R （5′-AACTGAATAGGCGTTAAGCTTC-3′） （Fig. 1）. Sequencing analysis showed that open reading frame （ORF） of the fragment was 1 170 bp （Fig .2）. Another DNA fragment of about 800 bp was PCR amplified by the same way， using the primers GhCHI-F （5′-CTTGCCTGCCTTAAACCTC-3′） and GhCHI-R （5′-TCATTTTGGGCTTCACTCA-3′）. Sequencing analysis showed that it had an ORF of 630 bp （Fig. 3）.
Physical and chemical properties of GhCHS and GhCHI proteins
Physical and chemical properties of GhCHS protein
The physical and chemical properties of GhCHS protein were analyzed using Protparam server. The results showed that GhCHS protein has a chemical formula of C1 900H3 045N507O563S19， a theoretical isoelectric point of about 6.12 and a molecular mass of about 42.608 kDa. As shown in Table 1， the protein is rich in Leu， Val， Ala， Gly， Glu and Lys. There are 48 negatively charged acidic amino acids （Glu and Asp） and 44 positively charged basic amino acids （Lys And Arg）， indicating that GhCHS protein is a negatively charged acidic protein. Its instability index is predicted to be 38.76. Its estimated half-life is about 30 h when expressed in vitro in mammalian reticulocytes， over 20 h when expressed in yeast cells， and 10 h when expressed in E. coli， indicating that it is a stable protein.
Physical and chemical properties of GhCHI protein
The physical and chemical properties of GhCHI protein were analyzed using Protparam server. The results showed that GhCHI protein has a chemical formula of C1 059H1 662N262O322S5， a theoretical isoelectric point of about 4.86 and a molecular mass of about 23.377 kDa. As shown in Table 2， the protein is rich in Glu， Val， Lys， Ala， Leu and Thr. There are 34 negatively charged acidic amino acids （Glu and Asp） and 22 positively charged basic amino acids （Lys And Arg）， indicating that GhCHS protein is a negatively charged acidic protein. Its instability index is predicted to be 35.39. Its estimated half-life is about 30 h when expressed in vitro in mammalian reticulocytes， over 20 h when expressed in yeast cells， and 10 h when expressed in E. coli， indicating that it is a stable protein. Hydrophobicity and subcellular localization of GhCHS and GhCHI proteins
Hydrophobicity and subcellular localization of GhCHS protein
The hydrophobicity of GhCHS protein was predicted using ProtScale （http：//web.expasy.org/protscale/）. As shown in Fig. 4， GhCHS protein has a grand average of hydropathicity （GRAVY） of -0.096， indicating that the entire protein is hydrophilic. The subcellular localization of GhCHS protein was predicted using PSORT. The results showed that the GhCHS protein may be located in the cytoplasm （with a score of 0.450）， peroxisome （with a score of score 0.300）， mitochondrial matrix （with a score of 0.100） and lysosome （with a score of 0.100） in cells. The presence of signal peptide of GhCHS protein was analyzed using SignalP 4.1 software， and the result showed there is no signal peptide sequence in GhCHS protein， so that the protein cannot pass through the cell membrane. The presence of transmembrane domain of the protein was predicted using TMHMM， and the results proved that there is no transmembrane helix in the protein. In summary， GhCHS protein is inferred to be a hydrophilic cytoplasmic protein.
Hydrophobicity and subcellular localization of GhCHI protein
The hydrophobicity of GhCHI protein was predicted using ProtScale （http：//web.expasy.org/protscale/）. As shown in Fig. 5， GhCHI protein has a grand average of hydropathicity （GRAVY） of -0.153， indicating that the entire protein is hydrophilic. The subcellular localization of GhCHS protein was predicted using PSORT， which revealed GhCHS protein may be located in the cytoplasm （with a score of 0.450）， peroxisome （with a score of 0.206）， lysosome （with a score of 0.100） and mitochondrial matrix （with a score of 0.100）. The presence of signal peptide of GhCHI protein was analyzed using SignalP 4.1 software， and the result showed there was no signal peptide sequence in this protein， indicating that it cannot pass through the cell membrane. TMHMM predicted that the protein has no transmembrane helix. Based on above results， GhCHI protein is a hydrophilic cytoplasmic protein.
Sequence alignment and phylogenetic analysis of GhCHS and GhCHI proteins
Sequence alignment and phylogenetic analysis of GhCHS protein
BlastP search revealed that the amino acid sequence of GhCHS of G. hirsutum has high similarity with that of other plant species. Then， amino acid sequences of the protein of G. hirsutum， Theobroma cacao （XP_007034442.1）， Hibiscus cannabinus （AIC75908.1）， Actinidia chinensis （AGV53049.1）， Camellia japonica （BAI66465.1）， Prunus avium （AJO67963.1）， Arabidopsis thaliana （NP_196897.1）， Solanum tuberosum （AEN83501.1）， Oryza sativa Japonica Group， （NP_001068009.1）， Pyrus pyrifolia （AFQ92052）. 1）， Rhus chinensis （AGH13332.1）， Nelumbo nucifera （ADD74167.1） and Malus domestica （BAB92996.1） were aligned using MEGA 5. The results showed that there is a high similarity in amino acid sequence of GhCHS protein between G. hirsutum and other plant species （Fig. 6）. Phylogenetic analysis indicated that GhCHS protein has the closest relationship with that of T. cacao （XP_007034442.1） （Fig. 7）. Sequence alignment and phylogenetic analysis of GhCHI protein
BlastP search revealed that the amino acid sequence of GhCHI of G. hirsutum had high similarity with that of other species. Then， amino acid sequences of the protein of G. hirsutum， T. cacao （XP_007011312.1）， H. cannabinus （AIC73814.1）， A. chinensis （AGV53050.1）， P. pyrifolia （AFQ92051.1）， Actinidia chrysantha （AIY53017.1）， Phyllanthus emblica （AHA61355.1）， Garcinia mangostana （ACM62743.1）， Lonicera japonica （AGE10598.1）， Lycium ruthenicum （AHH86093.1）， Arachis hypogaea （AEO17326.1）， Indosasa hispida （AHC07954.1）， Lycium chinense （AIC33515.1） and Morus alba （ALD83622.1） were aligned using MEGA 5.0. The results revealed that there is a high similarity in amino acid sequence of GhCHI protein between G. hirsutum and other plant species （Fig. 8）. Phylogenetic analysis indicated that GhCHI protein has the closest relationship with that of H. cannabinus （AIC73814.1） （Fig. 9）.
Phosphorylation sites of GhCHS and GhCHI proteins
Phosphorylation sites of GhCHS protein
Protein phosphorylation， a post-translational modification of proteins， participates in and regulates many cell activities. Phosphorylation alters the conformation， stability， activity and location of a protein in the cell[12]. GhCHS protein contains 24 threonine （Thr） and 22 serine （Ser） residues， which are potential phosphorylation sites. NetPhos 2.0 predicted that there are 17 potential phosphorylation sites in GhCHS protein including six Thr phosphorylation sites， nine Ser phosphorylation sites and two tyrosine （Tyr） phosphorylation sites （Fig. 10）， suggesting that GhCHS protein may be involved in kinase phosphorylation.
Phosphorylation sites of GhCHI protein
GhCHI protein contains 15 Thr and 10 Ser residues. NetPhos 2.0 predicted that there are eight potential phosphorylation sites in GhCHI protein including three Thr phosphorylation sites， three Ser phosphorylation sites and two tyrosine （Tyr） phosphorylation sites （Fig. 11）， suggesting that GhCHI protein may be involved in kinase phosphorylation.
Relative expression of GhCHS and GhCHI genes in G. hirsutum infected with V. dahliae VD07
The relative expression levels of GhCHS and GhCHI genes in true leaves of grafted G. hirsutum plants （7124 as the rootstock and HM-40 as the scion） were analyzed by qPCR. The results showed that the expression of GhCHS and GhCHI genes increased gradually over time after inoculation with V. dahliae VD07. The expression level of GhCHS gene on Day 2， 4， 6， 8 and 10 after the inoculation with V. dahliae VD07 was significantly higher than that on Day 0 （P<0.05）. The expression level of GhCHS gene on Day 10 was up-regulated by about 39 times， compared with that on Day 0. The expression level of GhCHI gene on Day 2， 4， 6， 8 and 10 after the inoculation with V. dahliae VD07 was also significantly higher than that on Day 0 （P<0.05）. The expression level of GhCHI gene on Day 10 was up-regulated by about 14 times， compared with that on Day 0. The results suggested that both GhCHS and GhCHI genes may play an important role in defense response of grafted cotton against Verticillium wilt. Function of GhCHS and GhCHI genes analyzed by VIGS
Sixteen days after the inoculation with A. tumefaciens transformed with pYL-156-pYL-192， pYL-156-GhCHS-pYL-192 or pYL-156-GhCHI-pYL -192， the total RNA of G. hirsutum leaves was extracted， reverse transcribed and subjected to quantitative and semi-quantitative PCR assays （Fig. 13）. The results showed that the expression levels of GhCHS and GhCHI in G. hirsutum leaves inoculated with VIGS vectors were decreased by 84.74% and 87.86%， respectively， compared with that in the leaves inoculated with empty vector （Fig. 14）. Twenty-five days after inoculation， the disease index of the grafted plants untransformed with A. tumefaciens was 31.2， indicating the plants were disease resistant； the disease index of the grafted plants transformed with empty vector was 30.0， indicating that the plants were disease resistant. The disease index of the grafted plants transformed with GhCHS VIGS vector was 72.5， and that in of the plants transformed with GhCHI VIGS vector was 67.5， indicating that the plants were disease susceptible （Fig. 15）.
Discussion
With development of plant genetic engineering， new technologies and bioinformatics， many genes of plants have been cloned， and their functions can be rapidly predicted and analyzed. VIGS is a fast and powerful method to study gene function in plants by silencing the gene of interest. Chalcone synthase and chalcone isomerase are the key enzymes in the synthesis of flavonoids， and involved in a variety of plant growth， stress and defense responses of plants. But their function in the resistance of cotton to Verticillium wilt has been rarely reported.
In this study， GhCHS and GhCHI genes were cloned from G. hirsutum， and analyzed bioinformatically. The results showed that both GhCHS and GhCHI proteins are hydrophilic cytoplasmic proteins， contains many phosphorylation sites， suggesting that they may be involved in kinase phosphorylation. In addition， both GhCHS and GhCHI genes are activated by the inoculation of V. dahliae， suggesting that they may be related to plant defense response. Using VIGS technology， we found that the silencing of either GhCHS or GhCHI gene lead to the loss of resistance to Verticillium wilt in cotton， which confirmed the function of the two genes in defense response against Verticillium wilt.
References
[1] WAKELYN PJ， N BERONIERE NR， FRENCH AD， et al. Cotton fiber chemistry and technology （International Fiber Science and Technology）[M]. USA： CRC Press， 2007. [2] BELL A A， HILLOCKS R J. Verticillium wilt.[M]. London： CAB International， 1992.
[3] ABDEL-LATEIF K， VAISSAYRE V， GHERBI H， et al. Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation[J]. New Phytologist， 2013， 199（4）： 1012-1021.
[4] MUIR SR， COLLINS GJ， ROBINSON S， et al. Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols[J]. Nature Biotechnology， 2001， 19， 470-474.
[5] BESSEAU S， LAURENT H， PIERRETTE G， et al. Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth[J]. The Plant Cell， 2007， 19： 148-162.
[6] KOES RE， FRANCESCA Q， JOSEPH NM. The flavonoid biosynthetic pathway in plants： Function and evolution[J]. Bioessays， 1994， 16： 123-132.
[7] CUI Y， FREDERIKSEN R， MAGILL C. Chalcone synthase and phenylalanine ammonialyase mRNA levels following exposure of sorghum seedlings to three fungal pathogens[J]. Physiological and Molecular Plant Pathology， 1996， 49（3）： 187-199.
[8] KAWASAKI T， KOEDUKA T， SUGIYAMA A， et al. Metabolic engineering of flavonoids with prenyltransferase and chalcone isomerase genes in tomato fruits[J]. Plant Biotechnology， 2014， 31（5）： 567-571.
[9] XU L， ZHU LF， TU LL， et al. Differential gene expressionin cotton defense response to Verticillium dahliae by SSH[J]. Journal of Phytopathology， 2011， 159（9）： 606-615.
[10] ROBERTSON D. Vigs vectors for gene silencing： Many targets， many tools[J]. Plant Biology， 2004， 55： 495-519.
[11] GAO XQ， JR RC B， SHAN LB， et al. Agrobacterium-mediated virus-induced gene silencing assay in cotton[J]. Journal of Visualized Experiments， 2011， 54： e2938.
[12] FANG ZZ， ZHOU DR， JIANG CC， et al. Cloning and bioinformatics analysis of Ran genes from Prunus salicina Lindl.[J]. Molecular Plant Breeding， 2014， 12（4）： 780-787.