摘要： 为了探讨花生几丁质酶在抵御真菌性病害中的作用，克隆了花生几丁质酶基因并通过遗传转化验证了该基因的功能。以花生品种花育23号基因组DNA和RNA为模板，通过PCR及RT-PCR扩增分别获得长度为1 779 bp的花生几丁质酶基因DNA序列及795 bp的全长cDNA序列。DNA及cDNA序列比对结果表明，该基因包含3个外显子和2个内含子，内含子剪切符合"GT……AG"规则，其cDNA序列被命名为Ah-Chi，编码265个氨基酸，GenBank注册号为HQ439775。利用NCBI在线Blast进行序列比对，结果表明，该蛋白产物与水稻、玉米、紫花苜蓿、大豆、拟南芥的相关蛋白的同源性分别达到83%，83%，72%，58%，49%。用Ah-Chi替换掉植物表达载体pCAMBIA1301上的Gus基因，成功构建了植物过表达载体pCAMBIA1301-Ah-Chi。利用农杆菌介导法将pCAMBIA1301-Ah-Chi转化花生胚小叶外植体，获得再生小苗，经嫁接移栽获得再生植株。利用PCR扩增筛选获得转基因阳性植株，RT-PCR扩增结果表明，转基因植株中Ah-Chi基因表达量增加。用黑斑病菌接种转基因植株和非转基因对照植株离体叶片7 d后，发现非转基因植株叶片褐化及坏死程度较为严重，而转基因植株叶片褐化及坏死程度相对较轻，初步说明转基因植株抗病性增强。

关键词: 花生, 几丁质酶基因, 遗传转化, 抗病性, 黑斑病菌

Abstract: The chitinase gene was cloned and its function was further analyzed by genetic transformation, aiming at exploring the role of chitinase gene in resisting fungal diseases in peanut.The DNA (1 779 bp) and cDNA (795 bp) of peanut chitinase gene were obtained successfully by PCR and RT-PCR amplification, with the genomic DNA and cDNA of peanut variety Huayu 23 as templates respectively.The sequence alignment result between DNA and cDNA showed that peanut chitinase gene contained three exons and two introns, conforming to "GT……AG" rule in intron splicing. The cDNA coding sequence contained 795 bp, coded for a 265-amino acid protein, and was named by Ah-Chi, which was registered in NCBI (GenBank accession No. HQ439775). As determined by Blast analysis, the Ah-Chi protein had homology with proteins from Oryza sativa (83%), Zea mays (83%), Medicago sativa (72%), Glycine max (58%) and Arabidopsis thaliana (49%). The over-expression vector pCAMBIA1301-Ah-Chi was constructed successfully by substituting Gus of pCAMBIA1301 for Ah-Chi, and then was transformed into peanut embryonic leaflets explants by Agrobacterium EHA105 mediated transformation. The regenerated plants were then obtained by grafting and transplanting those regenerated somatic embryo seedlings.These transgenic positive plants were further screened and verified by PCR amplification, and the increased expression level of Ah-Chi was confirmedin transgenic plants by RT-PCR amplification. Cercosporidium personatum was inoculated to the detached leaves of transgenic and non-transgenic plants. Seven days later, it was found that the non-transgenic plants showed more serious leaves' browning and necrosis than transgenic plants, which indicated that the disease resistance of transgenic plants were enhanced.

Key words: Peanut, Chitinase gene, Genetic transformation, Disease resistance, Cercosporidium personatum

中图分类号: 

• Q785