【作者】 张兵；

【导师】 张志宏； 马跃；

【作者基本信息】 沈阳农业大学 ， 果树学， 2017， 硕士

【摘要】 几丁质是真菌细胞壁重要的结构组成性物质,几丁质酶能够降解大多数病原真菌细胞壁中的几丁质,进而抑制真菌生长。几丁质酶抑制真菌生长的特点使它成为目前抗病基因工程育种研究的热点之一。本课题组前期对苹果转录组数据比对分析发现,抗病品种的几丁质酶基因的表达水平明显上调,因此本研究克隆苹果几丁质酶基因全长CDS序列,构建其过表达载体,并利用农杆菌介导的转化法获得苹果和拟南芥的转基因植株,为研究苹果抗病育种提供理论参考。主要研究结果如下:1.依据苹果几丁质酶基因的(MdChi)的序列,从苹果叶片中同源克隆得到MdChi基因全长CDS序列:该序列全长为954 bp,包含完整的开放阅读框,共编码317个氨基酸。生物信息学分析表明:MdChi编码的蛋白质的二级结构中存在大量的α-螺旋、无规则卷曲和少量扩展结构链,属于几丁质酶19家族,具有几丁质酶19家族特有的结构域:C端具有几丁质结合区,N端具有糖苷水解酶保守域。MdChi与已报道的其他植物的几丁质酶基因编码的氨基酸序列具有较高的同源性,与沙梨的同源性高达95%。2.以植物过表达载体pRI 101 AN为骨架,将MdChi全长CDS序列整合到pRI 101 AN载体上,构建苹果MsChi过表达载体,将其命名为pOX-MdChi。3.利用农杆菌介导的叶盘转化法,将MdChi基因导入到苹果中,获得4个苹果转基因植株。利用农杆菌介导的浸花转化法,将MdChi基因导入到拟南芥中,获得4个转基因植株。用灰霉病菌分别接种转基因植株的离体叶片,转基因植株的抗病性均强于对照,这表明MdChi基因的过量表达具有提高植物抗病能力的作用。更多还原

【Abstract】 Chitin is an important structural component of fungal cell wall.Chitinase can inhibit fungal growth by degrading chitin in the cell wall of pathogenic fungi.These featrures of chitinase make it one of the focuses of the current disease-resistance gene engineering breeding.We found that the expression level of chitinase gene in resistant cultivars was up-regulated significantly by comparing the apple transcriptome data.We cloned the full length CDS of MdChi gene from apple and constructed its overexpression vector.And then by use’ the method of Agrobacterium-mediated transformation,we obtained MdChi overexpression plants of apple and Arabidopsis thaliana.This study provided theoretical reference for the research of apple breeding.The main results are as follows:The whole CDS sequence of MdChi was cloned from leaves of apple based on.the chitinase gene sequence.The full CDS was 954 bp in size with an integrated open reading frame(ORF)encoding 317 amino acids;Bioinformatics analysis showed that the secondary structure of MdChi consists a lot of alpha helixes,random coils and a few extended strands and belonged to chitinase 19 family,containing the chitin-binding domain(CBD)in C-terminus and conseved domain of glycoside hydrolase in N-terminus;Amino acids of MdChi had high homology with other reported plants.which reached 95%with pear.The full length CDS of MdChi was intergrated into the plant expression vector pRI 101 AN and constructed the overexpression vector,which was named pOX-MdChi.Using the method of Agrobacterium-mediated transformation with the in vitro leaves as explants,the MdChi genes were introduced into apple.And four transgenic plants were obtained.The MdChi were introduced into A.thaliana by Agrobacterium-mediated transforma-tion.And four transgenic plants were obtained.The effect of transgenic plants on fungal resistance were stonger than control by inoculating the vitro leaves of transgenic plants with Botrytis cinerea,which suggested that overexpression of MdChi gene had a role in promoting the disease-resistance.更多还原