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为了开展基于青蒿发育特异及环境诱导转录物组学的新基因分离与鉴定,本研究采集经低温处理的盛花期青蒿(Artemisia annua)叶片提取总RNA,并进行全长cDNA表达序列标签(expres sedsequence tags,EST)克隆,经测序和同源性检索后提交GenBank.在已注册的75个序列中,共有4个全长cDNA与青蒿已知基因高度同源,其余71个EST在青蒿基因中无测序记录,但其中有34个序列与其他植物基因同源,包括24个已知蛋白编码序列和10个未知蛋白编码序列,另外37个序列在任何植物基因中均无测序记录,为首次克隆的植物新基因.为了研究青蒿基因对极端环境胁迫的响应模式,采用半定量PCR(semi-quantitative PCR,SQ-PCR)对冷处理前后青蒿试管苗中青蒿素合成基因ADS,CYP71AV1和CPR的表达水平进行了测定.结果显示,ADS和CYP71AV1基因受冷胁迫强烈诱导,但CPR基因未受明显影响.同时还发现这种冷胁迫诱导作用可被Ca2+通道抑制剂氯化镧(LaCl3)或Ca2+螯合剂EGTA所阻遏,表明Ca2+-CaM信号转导通路可能参与冷胁迫诱导ADS和CYP71AV1基因的表达.对冷胁迫青蒿试管苗的实时荧光定量PCR(real-time fluorescent quantitative PCR,RFQ-PCR)测定结果表明,CaM基因的表达水平上调2.5倍,从而印证了上述CaM介导信号转导与冷胁迫诱导基因表达之间相关的推论.利用RFQ-PCR对7个新分离青蒿EST进行了功能注释,结果显示,D/LTSRP,UBE,AR/DAP和POD1基因的冷胁迫诱导表达水平分别上调约8.0,5.0,2.3和1.5倍,而CHI和RGP基因的表达无明显上调或下调.本研究首次在转录水平上对青蒿素合成基因及青蒿新EST的冷胁迫诱导表达模式进行了初步探讨,有助于深入了解青蒿素合成与累积的内在规律及其协同调节机制,为促进青蒿的遗传性状改良及代谢工程指导的育种工作打下基础.
In order to isolate and identify new genes based on Artemisia annua development and environment-induced transcriptomics, total RNA was extracted from leaves of Artemisia annua at low temperature and the full-length cDNA sequence tagging was performed expres sedsequence tags (ESTs)) were cloned and sequenced. The sequences were submitted to GenBank for homology search. Among the 75 registered sequences, four full-length cDNAs were highly homologous to the known genes of Artemisia annua L. The remaining 71 ESTs Artemisia annua gene without sequencing records, but there are 34 sequences homologous to other plant genes, including 24 known protein coding sequence and 10 unknown protein coding sequence, the other 37 sequences in any plant gene without sequencing records, Which is the first plant cloning gene.In order to study the response mode of Artemisia annua L. to extreme environmental stress, semi-quantitative PCR (SQ-PCR) was used to detect the expression of artemisinin synthesis genes ADS, CYP71AV1 and CPR.The results showed that the genes of ADS and CYP71AV1 were strongly induced by cold stress but the CPR gene was not significantly affected.It was also found that this cold stress induced by cold stress could be induced by Ca2 + Inhibitor LaCl3 or Ca2 + chelator EGTA, indicating that Ca2 + -CaM signal transduction pathway may be involved in the expression of ADS and CYP71AV1 gene under cold stress.According to real-time fluorescent quantitative PCR time fluorescence quantitative PCR (RFQ-PCR) assay showed that the expression level of CaM gene was up-regulated by 2.5-fold, confirming the corollary of the above correlation between CaM-mediated signal transduction and cold stress-induced gene expression. Seven newly isolated ESTs from A. annua were functional annotated and the results showed that cold stress-induced expression levels of D / LTSRP, UBE, AR / DAP and POD1 genes were increased by about 8.0, 5.0, 2.3 and 1.5 times, respectively, whereas CHI and RGP genes The expression of artemisinin and the new EST induced by cold stress induced expression of a preliminary study will help to understand the depth of artemisinin synthesis and accumulation of the intrinsic The rules and their synergistic regulation mechanism lay the foundation for promoting the genetic improvement of A. annua and the breeding work guided by metabolic engineering.