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植物类受体蛋白激酶(Receptor-like protein kinase,RLK)是蛋白激酶的一个亚家族,参与植物的信号转导,在植物生长发育、抗病等过程中起重要作用。在前期研究中自二倍体马铃薯高抗青枯病基因型ED13中获得了类受体蛋白激酶基因StRLK。利用StRLK基因的特异区段,以pUCCRNAi为中间克隆载体、pCHF1为植物表达载体,构建了该基因的RNA干扰载体pCHF1-StRLK。进一步以ED13的茎段为外植体,利用农杆菌介导法将pCHF1-StRLK导入ED13中,获得了5株再生植株。用CaMV35S启动子特异引物对5株再生植株进行PCR扩增,均得到大小约500 bp的特异性条带,初步证明pCHF1-StRLK成功转入ED13。以ED13为对照,利用StRLK基因的特异引物对这5株再生植株进行半定量RT-PCR分析,结果表明,该基因的表达在5个转基因植株中均受到了不同程度的抑制,说明导入的pCHF1-StRLK发挥了干扰活性,且干扰效果与基因的插入位点有关,为进一步研究StRLK基因的功能提供依据。
Receptor-like protein kinase (RLK) is a subfamily of protein kinases involved in plant signal transduction and plays an important role in plant growth and disease resistance. In the previous study, the receptor-like protein kinase gene StRLK was obtained from the diploid potato high resistant bacterial wilt genotype ED13. Using the specific region of StRLK gene, pUCCRNAi was used as an intermediate cloning vector and pCHF1 as plant expression vector. The RNA interference vector pCHF1-StRLK was constructed. Further, using the stem segment of ED13 as explant, pCHF1-StRLK was introduced into ED13 by Agrobacterium-mediated method, and 5 regenerated plants were obtained. Specific amplification of 500 bp was obtained by PCR amplification of 5 regenerated plants with CaMV35S promoter-specific primers. It was initially proved that pCHF1-StRLK was successfully transferred into ED13. The results of semi-quantitative RT-PCR analysis showed that the expression level of this gene was inhibited in all five transgenic plants by using the specific primers of StRLK gene in ED13, indicating that the introduced pCHF1 -StRLK exerted the interference activity, and the interference effect was related to the gene insertion site, which provided the basis for further study on the function of StRLK gene.