本文關(guān)鍵詞： 大眼長蝽 卵黃原蛋白 基因克隆 37KDa蛋白 生長繁殖　出處：《中國農(nóng)業(yè)科學(xué)院》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：大眼長蝽Geocoris pallidipennis是一種重要的捕食性天敵昆蟲,是棉花、煙草等作物害蟲的主要天敵,在害蟲生物防治的應(yīng)用中具有廣闊前景。近年來,對大眼長蝽的研究主要集中在形態(tài)特征,生活習(xí)性等生態(tài)學(xué)和捕食功能上,對其繁殖關(guān)鍵因子——卵黃原蛋白的研究未見報道。本文克隆了大眼長蝽卵黃原蛋白基因,研究了大眼長蝽卵黃原蛋白基因的分子特征,同時本研究表達了37KDa蛋白,明確了37KDa蛋白對大眼長蝽生長繁殖的影響,為人工飼料的改進和天敵昆蟲的大規(guī)模繁殖提供了理論基礎(chǔ)和相關(guān)技術(shù)。本論文研究結(jié)果如下:1.利用RT-PCR和RACE技術(shù)克隆得到了大眼長蝽卵黃原蛋白(Vg)基因全序列。該基因c DNA全長5667bp(Gen Bank登錄號:KP688587),編碼1848個氨基酸殘基,N-末端的前19個氨基酸為信號肽。序列分析顯示該卵黃原蛋白序列具有昆蟲卵黃原蛋白所特有的保守基序和RXXR酶切位點。編碼的氨基酸序列比對顯示與半翅目昆蟲Vg氨基酸序列相似度較高,表明克隆的c DNA序列是大眼長蝽的Vg基因序列。2.ELISA檢測發(fā)現(xiàn)隨著發(fā)育時間的延長,卵黃原蛋白表達量逐漸增加,羽化后22天達到高峰,隨后開始下降。本研究利用已克隆的大眼長蝽卵黃原蛋白基因序列,設(shè)計特異引物擴增目的基因片段,成功構(gòu)建了Vg基因片段重組表達載體,并進一步轉(zhuǎn)化寄主細(xì)胞,在不同條件下誘導(dǎo)Vg基因目的片段在大腸桿菌菌株中穩(wěn)定表達,通過Ni-NTA柱分離純化出目的蛋白。3.研究了Vg基因編碼的37KDa蛋白對大眼長蝽生長發(fā)育和繁殖的影響,明確了該蛋白在大眼長蝽中的生理功能。本研究表達的37KDa蛋白飼喂大眼長蝽結(jié)果表明:目的蛋白飼喂大眼長蝽可以顯著降低其五齡發(fā)育和產(chǎn)卵前期,并顯著提高產(chǎn)卵量;同時卵的孵化率與兩種對照相比分別提高了18.95%和15.92%。研究結(jié)果表明37KDa蛋白具有顯著促進大眼長蝽發(fā)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.
【學(xué)位授予單位】：中國農(nóng)業(yè)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S476.2

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