大眼长蝽卵黄原蛋白基因克

发布时间：2018-02-24 14:09

本文关键词： 大眼长蝽卵黄原蛋白基因克隆 37KDa蛋白生长繁殖　出处：《中国农业科学院》2015年硕士论文　论文类型：学位论文

【摘要】：大眼长蝽Geocoris pallidipennis是一种重要的捕食性天敌昆虫,是棉花、烟草等作物害虫的主要天敌,在害虫生物防治的应用中具有广阔前景。近年来,对大眼长蝽的研究主要集中在形态特征,生活习性等生态学和捕食功能上,对其繁殖关键因子——卵黄原蛋白的研究未见报道。本文克隆了大眼长蝽卵黄原蛋白基因,研究了大眼长蝽卵黄原蛋白基因的分子特征,同时本研究表达了37KDa蛋白,明确了37KDa蛋白对大眼长蝽生长繁殖的影响,为人工饲料的改进和天敌昆虫的大规模繁殖提供了理论基础和相关技术。本论文研究结果如下:1.利用RT-PCR和RACE技术克隆得到了大眼长蝽卵黄原蛋白(Vg)基因全序列。该基因c DNA全长5667bp(Gen Bank登录号:KP688587),编码1848个氨基酸残基,N-末端的前19个氨基酸为信号肽。序列分析显示该卵黄原蛋白序列具有昆虫卵黄原蛋白所特有的保守基序和RXXR酶切位点。编码的氨基酸序列比对显示与半翅目昆虫Vg氨基酸序列相似度较高,表明克隆的c DNA序列是大眼长蝽的Vg基因序列。2.ELISA检测发现随着发育时间的延长,卵黄原蛋白表达量逐渐增加,羽化后22天达到高峰,随后开始下降。本研究利用已克隆的大眼长蝽卵黄原蛋白基因序列,设计特异引物扩增目的基因片段,成功构建了Vg基因片段重组表达载体,并进一步转化寄主细胞,在不同条件下诱导Vg基因目的片段在大肠杆菌菌株中稳定表达,通过Ni-NTA柱分离纯化出目的蛋白。3.研究了Vg基因编码的37KDa蛋白对大眼长蝽生长发育和繁殖的影响,明确了该蛋白在大眼长蝽中的生理功能。本研究表达的37KDa蛋白饲喂大眼长蝽结果表明:目的蛋白饲喂大眼长蝽可以显著降低其五龄发育和产卵前期,并显著提高产卵量;同时卵的孵化率与两种对照相比分别提高了18.95%和15.92%。研究结果表明37KDa蛋白具有显著促进大眼长蝽发育和繁殖的生理功能。
[Abstract]:Geocoris pallidipennis is an important predatory natural enemy insect, which is the main natural enemy of cotton, tobacco and other crop pests. The study of vitellogenin, the key reproductive factor of vitellogenin, has not been reported in ecology and predation function. In this paper, the gene of vitellogenin was cloned and the molecular characteristics of egg yolk protein gene were studied. At the same time, 37KDa protein was expressed in this study, and the effect of 37KDa protein on the growth and reproduction of stink bug was clarified. The research results are as follows: 1. Using RT-PCR and RACE techniques, the whole sequence of egg yolk protein (Vg) gene was obtained. The full length of c DNA is 5667bpnGen Bank accession number: KP688587N, encoding the first 19 amino acids at the N-terminal of 1848 amino acid residues are signal peptides. Sequence analysis shows that the egg yolk protein sequence has the conserved motif and RXXR characteristic of insect vitellogenin. The amino acid sequence alignment showed that the amino acid sequence was similar to the Vg amino acid sequence of Hemiptera insects. The results showed that the cloned c DNA sequence was the Vg gene sequence of the stink bug. 2. Elisa showed that the expression of egg yolk protein increased with the development time, and reached the peak at 22 days after emergence. In this study, specific primers were designed to amplify the target gene fragment, and the recombinant expression vector of VG gene fragment was successfully constructed, and further transformed into host cells. Under different conditions, the target fragment of VG gene was induced to express stably in Escherichia coli strain, and the target protein .3was purified by Ni-NTA column. The effect of 37KDa protein encoded by Vg gene on the growth, development and reproduction of the bug was studied. The physiological function of the protein was clarified. The 37KDa protein was expressed in this study. The results showed that the feeding of the protein could significantly reduce the fifth instar development and pre-oviposition, and increase the amount of oviposition. The hatching rate of eggs was increased by 18.95% and 15.92, respectively. The results showed that 37KDa protein had the physiological function of promoting the development and reproduction of stink bug.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.2

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