【摘要】：由Bt(Bacillus thuringiensis)產(chǎn)生的Cry蛋白引起昆蟲死亡的過程中,Cry蛋白與昆蟲中腸受體蛋白的結合起著至關重要的作用,結合能力的下降或喪失將導致昆蟲對Cry蛋白抗性的產(chǎn)生。已有報道表明Polycalin是昆蟲中腸上Cry1Ac的結合蛋白,本文在棉鈴蟲Polycalin基因的克隆、m RNA表達譜分析及其功能方面進行了研究。通過PCR結合RACE技術克隆獲得了棉鈴蟲Polycalin基因的全長序列;利用實時熒光定量PCR技術測定了在棉鈴蟲不同發(fā)育時期、幼蟲腸道不同部位的Polycalin基因表達量;比較了棉鈴蟲取食含Cry1Ac蛋白的飼料后,Polycalin基因表達量的變化;利用原核表達的方法表達Polycalin的部分片段,合成抗體后,通過western blot和ligand blot的方法檢測了Polycalin蛋白與Cry1Ac的結合特性;運用PCR擴增、熒光定量和i TRAQ的方法,比較了抗性和敏感棉鈴蟲Polycalin在基因序列、m RNA表達量、蛋白表達量方面的差異。主要結果如下:1.通過PCR結合RACE技術克隆獲得了棉鈴蟲Polycalin基因的全長序列,該基因全長序列為2955bp,開放閱讀框為2781bp,編碼926個氨基酸(Gen Bank登錄號為KP100652);預測蛋白的分子量為101.68KD,等電點為4.57。推導的氨基酸序列中,N末端含有20個氨基酸組成的信號肽,含有8個O-糖基化位點,3個N-糖基化位點,C末端存在2個GPI結合位點。2.Polycalin在棉鈴蟲所有發(fā)育階段都可以表達,幼蟲期表達量較高,尤其在1-3齡幼蟲體內(nèi)表達量最高,在卵、成蟲和蛹中的表達量較低。棉鈴蟲四齡幼蟲取食含活化Cry1Ac蛋白的人工飼料后,Polycalin基因的表達受到抑制。3.通過原核表達Polycalin的部分片段,合成抗體后,通過western blot和ligand blot的方法檢測了Polycalin蛋白與Cry1Ac的結合特性�？剐约懊舾忻掴徬x品系Polycalin基因序列在編碼區(qū)核苷酸水平上存在4個突變,但是在氨基酸水平上沒有差異;抗性品系4齡棉鈴蟲幼蟲Polycalin基因的表達量極顯著高于敏感品系,敏感品系棉鈴蟲2齡幼蟲及成蟲中Polycalin基因的表達量都顯著高于抗性品系。Western blot及i TRAQ實驗結果表明,Polycalin蛋白在抗性品系中的表達量都比較高。以上結果說明棉鈴蟲Polycalin是Cry1Ac的結合蛋白,并且在抗性品系中Polycalin的蛋白的表達量升高。Polycalin基因是否在棉鈴蟲的抗性演化中是否發(fā)揮作用還需要進一步的驗證。
[Abstract]:In the process of insect death caused by Cry protein produced by Bt (Bacillus thuringiensis), the binding of Cry protein to insect midgut receptor protein plays an important role. The decrease or loss of binding ability will lead to the production of resistance to Cry protein in insects. It has been reported that Polycalin is the binding protein of Cry1Ac in the midgut of insects. In this paper, we studied the expression profile and function of Polycalin gene clone of Helicoverpa armigera. The full-length sequence of Polycalin gene of Helicoverpa armigera was cloned by PCR combined with RACE, and the expression of Polycalin gene in different parts of larval intestine of Helicoverpa armigera was determined by real-time fluorescence quantitative PCR. The changes of Polycalin gene expression in Helicoverpa armigera fed with Cry1Ac protein were compared, and the partial Polycalin fragment was expressed by prokaryotic expression method. After the antibody was synthesized, the binding characteristics of Polycalin protein to Cry1Ac were detected by western blot and ligand blot methods. The differences of gene sequence, m RNA expression and protein expression between resistant and sensitive Polycalin of Helicoverpa armigera were compared by PCR amplification, fluorescence quantitative analysis and I TRAQ. The main results are as follows: 1. The full-length Polycalin gene of Helicoverpa armigera was cloned by PCR combined with RACE. The full-length sequence of Polycalin gene is 2955 BP, the open reading frame is 2781 BP and encodes 926 amino acid (Gen Bank accession number KP100652. The predicted molecular weight of the protein was 101.68 KD and the isoelectric point was 4.57. The deduced amino acid sequence contained 20 amino acid signal peptides, 8 O-glycosylation sites and 3 N-glycosylation sites. 2. Polycalin can be expressed in all developmental stages of Helicoverpa armigera, especially in 1st instar larvae, and the expression of Polycalin in eggs, adults and pupae is low. 2. Polycalin can be expressed in all developmental stages of Helicoverpa armigera, especially in 1st instar larvae. The expression of Polycalin gene in the fourth instar larvae of Helicoverpa armigera was inhibited by feeding artificial diet containing activated Cry1Ac protein. 3. A partial fragment of Polycalin was expressed in E. coli. After the antibody was synthesized, the binding properties of Polycalin protein to Cry1Ac were detected by western blot and ligand blot. There were 4 mutations in nucleotide level of Polycalin gene in resistant and sensitive strains of Helicoverpa armigera, but there was no difference in amino acid level. The expression of Polycalin gene in 4th instar larvae of Helicoverpa armigera was significantly higher than that of susceptible strain, and the expression of Polycalin gene in 2nd instar larvae and adults of cotton bollworm was significantly higher than that of resistant strain. Western blot and I TRAQ test showed that the expression of Polycalin gene was significantly higher than that of resistant strain. The expression of Polycalin protein was higher in resistant lines. The above results suggest that Polycalin of Helicoverpa armigera is the binding protein of Cry1Ac, and the expression of Polycalin protein in resistant strain is increased. Whether Polycalin gene plays a role in the resistance evolution of Helicoverpa armigera needs further verification.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S433

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本文編號：2452033