稻纵卷叶螟几丁质合成酶及合成通路相关酶基因的鉴定及表达分析

Identification and expression analysis of chitin synthase and related enzymes in the chitin biosynthetic pathway genes of Cnaphalocrocis medinalis

【目的】稻纵卷叶螟Cnaphalocrocis medinalis(Guenee)是水稻上的四大害虫之一,危害较为严重,近年来以几丁质合成和代谢过程作为害虫防治的标靶研究已成为热点。为阐明几丁质合成酶及合成通路上关键酶的作用,本研究开展了对稻纵卷叶螟几丁质合成酶及合成相关通路上关键酶的克隆及时空表达分析。【方法】本研究基于稻纵卷叶螟转录组,结合PCR及RACE技术,克隆了几丁质合成酶代谢通路上的4条基因的c DNA全长;利用生物信息学软件对序列进行结构预测、序列比对和进化分析;采用实时定量PCR技术研究了4条基因在不同虫态和幼虫的不同组织中的表达情况。【结果】获得了2条几丁质合成酶序列及2条合成通路上的基因序列,包括几丁质合成酶A(Chitin synthase A,CHSA),几丁质合成酶B(Chitin synthase B,CHSB),N-乙酰葡糖胺磷酸变位酶(Phosphoacetylglucosamine mutase,PGM和UDP-N-乙酰葡萄糖焦磷酸化酶(UDP-N-acetylglucosamine pyrophosphorylase,UAP),并分别命名为Cm CHSA、Cm CHSB、Cm PGM和Cm UAP;序列分析显示Cm CHSA序列全长4 868 bp,编码1 564个氨基酸。Cm CHSB序列全长4 651 bp,编码1 525个氨基酸。Cm PGM全长1 934 bp,编码548个氨基酸。Cm UAP序列全长1 837 bp,编码487个氨基酸。实时定量研究表明,Cm UAP和Cm PGM在血淋巴中表达量最高,Cm CHSA在头部和表皮中表达量较高,而Cm CHSB在中肠中表达量最高。【结论】本研究得到了稻纵卷叶螟几丁质合成路径的4个关键酶基因c DNA全长,它们在稻纵卷叶螟的不同组织和虫态中呈现了差异显著的时空表达,本文为进一步探究稻纵卷叶螟的几丁质合成酶的生理功能和几丁质的合成代谢途径奠定了基础。

[Objectives] The rice leaf folder, Cnaphalocrocis medinalis（Guenee）, is one of four rice pest insects that cause serious crop damage. In recent years, chitin synthesis and metabolism has become a focus of pest control research. Cloning and spatio-temporal expression of two chitin synthases, and other two key enzymes in the chitin biosynthetic pathway encoding genes in C. medinalis, were conducted to reveal the function of these genes. [Methods] Based on transcriptome data, we used the PCR and RACE techniques to clone the full length c DNA sequences of 4 key enzymes in the chitin biosynthetic pathway. Prediction of the structure, sequence alignment and phylogenetic analysis of the products of these 4 genes were performed using different bioinformatics software. The relative expression levels of the 4 genes in different developmental stages and larval tissues of C. medinalis were determined with quantitative Real-time PCR. [Results] Two full-length c DNA sequences encoding chitin synthase, and two full-length c DNA sequences encoding other two key enzymes related to the chitin biosynthetic pathway, were obtained. These were; Chitin Synthase A（CHSA）, Chitin Synthase B（CHSB）, Phosphoacetylglucosamine Mutase（PGM） and UDP-N-acetylglucosamine pyrophosphorylase（UAP）（hereafter Cm CHSA, Cm CHSB, Cm PGM and Cm UAP, respectively）. Sequence analysis shows that the full length of the Cm CHSA gene is 4 868 bp, which encodes a polypeptide of 1 564 amino acids, the full length of the Cm CHSB gene is 4 651 bp, which encodes a polypeptide of 1 525 amino acids, the full length of Cm PGM gene is 1 934 bp, which encodes a polypeptide of 548 amino acids, and the full length of Cm UAP gene is 1 837 bp, which encodes a polypeptide of 487 amino acids. The results of RT-q PCR indicate that Cm UAP and Cm PGM had higher expression in hemolymph, whereas Cm CHSA was more highly expressed in the head and integument than the midgut and Cm CHSB was more highly expressed in the midgut than in other tissues. [Conclusion] Four key chitin biosynthetic pathway genes were cloned in C. medinalis. These have significantly different spatio-temporal expression patterns in different developmental and tissues. This work lays a foundation for future research on the function of Cm CHS and the chitin biosynthetic pathway.