黄瓜几丁质酶基因克隆及与白粉病抗性关系的初步研究

A Preliminary Study on Cloning of Chitinase Gene and Relationships to Resistance of Powdery Mildew in Cucumber(Cucumis sativus L.)

本研究采用常规PCR技术在黄瓜抗白粉病品种JIN5-508中克隆得到几丁质酶基因,并利用荧光定量PCR分析该基因在接种白粉菌后的表达变化。研究结果显示:接菌后几丁质酶基因表达量明显上升,在24 h达到最高值,是其对照的7倍;接菌24 h后,几丁质酶基因表达量迅速下降,最终与对照趋于一致。从具有不同抗白粉病特性的品种中克隆得到几丁质酶基因并进行同源性比对,获得4个多态性位点,对品种间几丁质酶基因开放阅读框(ORF)所编码的氨基酸序列进行了比对,发现了2个氨基酸差异位点,其中抗感白粉病品种在第557个碱基位点即第184个氨基酸位点上存在差异。进一步以抗病品种JIN5-508与感病品种D8杂交得到的F2代为材料,从苗期(2~3片真叶)人工接种白粉菌,1周后根据幼苗发病情况鉴定出F2代中表现为抗病的植株和感病的植株,并以此为材料克隆几丁质酶基因,成功克隆得到F2抗与F2感的几丁质酶基因,发现F2抗性植株中克隆到的几丁质酶基因差异氨基酸位点处的氨基酸与抗病品种一致,而从F2感病株中克隆得到的几丁质酶基因差异氨基酸位点处的氨基酸与感病品种一致。研究结果表明,几丁质酶基因与黄瓜的白粉病抗性密切相关,可利用此差异位点进一步发展与黄瓜白粉病相关的功能性分子标记。

Chitinase gene was cloned in cucumber variety'JIN5-508'through PCR amplification. Transcript abudances of the chitinase gene after inoculation with Powdery Mildew showed that the peak expression level is24 h after infection, about 7 times of control in 24 h, then decreased gradually. Four SNPS in chitinase gene were also found among different resistant varieties through nucleotide sequence alignment ananlysis, including two amino acid mutations. The differences between resistant and susceptible varieties are mainly in the number of 557nucleotide(the 184 th amino acid) of the sequence. An F2 population derived from a cross between JIN5-508 and D8 were used to infect with powdery mildew spores at 2~3 true leaves stages. Assessment of resistant ability plants were performed after 1 week of infection, and chitinase genes were successfully cloned from two groups of plants.We found the mutant amino acid in resistant plants was consistent with resistant varieties JIN5-508 and Ningyang6, the mutant amino acid in susceptible plants was consistent with susceptible varieties D8 and Yihuang.These results showed that chitinase gene might be related to powdery mildew resistance of cucumber, the mutantsite identified can be used to develop functional markers that associated with cucumber powdery mildew resistance.