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通过农杆菌介导法将杜仲几丁质酶基因EuCHIT1遗传转化‘Micro-Tom’番茄,经筛选和PCR鉴定获得38株转基因植株。对野生型和转EuCHIT1番茄几丁质酶活性、灰霉病抗性、保护酶活性以及病程相关蛋白基因表达水平等进行分析,结果表明,转EuCHIT1番茄植株几丁质酶活性为2 059.48 U·g~(-1)(FW),较野生型高62.14%,差异达到极显著水平。对野生型和转EuCHIT1番茄植株接种灰霉菌后,转EuCHIT1番茄发病时间比野生型推迟3 d。分别对未接种和接种灰霉菌6 d后的野生型和转EuCHIT1番茄SOD、POD、CAT活性和MDA含量分析表明,接菌前,转EuCHIT1番茄植株中POD活性为2 825.85 U·g~(-1)(FW),比野生型高48.63%;MDA含量为21.84 nmol·g~(-1)(FW),比野生型低28.25%;而SOD和CAT活性与野生型相比无显著差异。接菌后,转EuCHIT1番茄植株中SOD、POD和CAT活性分别为510.44、5 423.92和603.59 U·g~(-1)(FW),分别比野生型高19.48%、116.08%和53.80%;MDA含量为26.49 nmol·g~(-1)(FW),比野生型低37.65%。说明EuCHIT1基因表达增强了番茄植株的抗氧化能力,从而减少灰霉病对番茄的损伤。病程相关蛋白基因表达分析表明,接菌前,转EuCHIT1番茄中PR~(-1)a、PR-2和PR-5基因表达量均高于野生型,分别为野生型的2.23、11.69和1.80倍,PR-NP24表达量与野生型无显著差异;接菌后,转EuCHIT1番茄的PR~(-1)a、PR-2、PR-NP24和PR-5表达量均极显著高于野生型,分别是野生型的4.35、10.37、16.58和5.02倍。上述研究结果说明,转EuCHIT1基因番茄提高对灰霉病抗性,与保护性酶活性提高以及病程相关蛋白基因表达上调有关。
Euchit1, an Eucommia ulmoides chitinase gene, was transformed into ’Micro-Tom’ tomato by Agrobacterium-mediated transformation. Thirty-eight transgenic plants were obtained by screening and PCR. Analysis of chitinase activity, gray mold resistance, protective enzyme activity and expression of disease-related protein genes in wild-type and transgenic EuCHIT1 tomato showed that the activity of chitinase in EuCHIT1 tomato plants was 2 059.48 U · g ~ (-1) (FW), 62.14% higher than the wild type, the difference reached extremely significant level. After inoculation of Botrytis cinerea on wild type and EuCHIT1 tomato plants, the onset time of EuCHIT1 tomato was delayed 3 days than that of wild type. The activities of SOD, POD, CAT and the content of MDA in wild and transgenic tomato plants of EuCHIT1 which were not inoculated and inoculated with Botrytis cinerea for 6 d respectively showed that the activity of POD in tomato plants before transferring to inoculation was 2 825.85 U · g ~ (- 1) (FW), which was 48.63% higher than that of the wild type. MDA content was 21.84 nmol · g -1 (FW), which was 28.25% lower than that of the wild type. However, SOD and CAT activities were not significantly different from those of the wild type. After inoculation, the activities of SOD, POD and CAT in tomato plants that transferred EuCHIT1 were 510.44, 523.92 and 603.59 U · g -1, respectively, which were 19.48%, 116.08% and 53.80% higher than that of wild type respectively. MDA, The content was 26.49 nmol · g -1 (FW), which was 37.65% lower than the wild type. This indicated that EuCHIT1 gene expression enhanced the antioxidant capacity of tomato plants and thus reduced tomato damage caused by Botrytis cinerea. The gene expression analysis of disease-related proteins showed that the expression levels of PR-1, PR-2 and PR-5 in EuCHIT1 tomato before inoculation were higher than those in wild type, which were 2.23, 11.69 and 1.80 PR-NP24, PR-NP24 and PR-5 were significantly higher than those of wild-type , Respectively, 4.35,10.37,16.58 and 5.02 times of the wild type. The results of the above study showed that tomato with EuCHIT1 gene transfer increased resistance to Botrytis cinerea, which was related to the increase of protective enzyme activity and the up-regulation of gene expression of disease-related proteins.