棉铃虫Polycalin基因的克

发布时间：2019-04-02 00:06

【摘要】：由Bt(Bacillus thuringiensis)产生的Cry蛋白引起昆虫死亡的过程中,Cry蛋白与昆虫中肠受体蛋白的结合起着至关重要的作用,结合能力的下降或丧失将导致昆虫对Cry蛋白抗性的产生。已有报道表明Polycalin是昆虫中肠上Cry1Ac的结合蛋白,本文在棉铃虫Polycalin基因的克隆、m RNA表达谱分析及其功能方面进行了研究。通过PCR结合RACE技术克隆获得了棉铃虫Polycalin基因的全长序列;利用实时荧光定量PCR技术测定了在棉铃虫不同发育时期、幼虫肠道不同部位的Polycalin基因表达量;比较了棉铃虫取食含Cry1Ac蛋白的饲料后,Polycalin基因表达量的变化;利用原核表达的方法表达Polycalin的部分片段,合成抗体后,通过western blot和ligand blot的方法检测了Polycalin蛋白与Cry1Ac的结合特性;运用PCR扩增、荧光定量和i TRAQ的方法,比较了抗性和敏感棉铃虫Polycalin在基因序列、m RNA表达量、蛋白表达量方面的差异。主要结果如下:1.通过PCR结合RACE技术克隆获得了棉铃虫Polycalin基因的全长序列,该基因全长序列为2955bp,开放阅读框为2781bp,编码926个氨基酸(Gen Bank登录号为KP100652);预测蛋白的分子量为101.68KD,等电点为4.57。推导的氨基酸序列中,N末端含有20个氨基酸组成的信号肽,含有8个O-糖基化位点,3个N-糖基化位点,C末端存在2个GPI结合位点。2.Polycalin在棉铃虫所有发育阶段都可以表达,幼虫期表达量较高,尤其在1-3龄幼虫体内表达量最高,在卵、成虫和蛹中的表达量较低。棉铃虫四龄幼虫取食含活化Cry1Ac蛋白的人工饲料后,Polycalin基因的表达受到抑制。3.通过原核表达Polycalin的部分片段,合成抗体后,通过western blot和ligand blot的方法检测了Polycalin蛋白与Cry1Ac的结合特性。抗性及敏感棉铃虫品系Polycalin基因序列在编码区核苷酸水平上存在4个突变,但是在氨基酸水平上没有差异;抗性品系4龄棉铃虫幼虫Polycalin基因的表达量极显著高于敏感品系,敏感品系棉铃虫2龄幼虫及成虫中Polycalin基因的表达量都显著高于抗性品系。Western blot及i TRAQ实验结果表明,Polycalin蛋白在抗性品系中的表达量都比较高。以上结果说明棉铃虫Polycalin是Cry1Ac的结合蛋白,并且在抗性品系中Polycalin的蛋白的表达量升高。Polycalin基因是否在棉铃虫的抗性演化中是否发挥作用还需要进一步的验证。
[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.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433

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