【作者】 杜恩岐；

【导师】 齐义鹏；

【作者基本信息】 武汉大学 ， 微生物学， 2006， 博士

【摘要】 p13基因由我们实验室于1995年首次报道。随着大规模基因组测序的发展,越来越多的杆状病毒全基因组被测定,目前已报道的杆状病毒p13基因多达二十多个。奇怪地是这些p13基因均特异地存在于II型核型多角体病毒和部分颗粒体病毒的基因组中。而I型核型多角体病毒基因组中则不存在。对于p13基因的启动子和编码区的结构功能目前已有预测,但尚无相关研究工作报道。本研究对p13基因的启动子活性、p13基因的进化、在天然系统及异源系统中的功能及其潜在的杀虫活性进行了研究,为p13基因进一步深入研究和杀虫潜力应用的开创之举。目前已有14类不同杆状病毒的22个p13基因序列报道,我们经DNASTAR软件对已报道的14类杆状病毒(每类各选一个序列)P13氨基酸同源性比较发现,不同杆状病毒P13之间的同源性在42.1%-74.7%,其中Ls-P13与HaNPV P13同源性最高(58.7%)与PoGV P13最低(44%)。对P13的氨基酸同源性比较和进化树分析表明P13可被分为两个群,I群为GV的P13,它们之间有高度同源性。II群为II型NPV的P13,它们之间同源性差异较大。其中II群根据同源性又可分为两个亚群:I亚群为LsNPV,HaNPV和SlNPV三种,其他II型NPVs的P13为第II亚群。p13基因5’UTR普遍存在早期启动子核心序列GTGTTATA及CAT/AT盒和晚期启动子核心序列TTAAG盒。在早晚期启动子之间有一3-30aa组成的小ORF,推测这一小顺反子可能具有重要的调控功能。在p13基因5’UTR的上游,有一个杆状病毒普遍存在的同源重复序列(Homologue repeat,简称hr),一般hr有多个,而p13基因的hr的数目因病毒种类的不同而不同。已经证明,杆状病毒的hr是DNA复制的ori和转录激活的增强子,对早期基因的表达有增强作用。因此由p13基因的5’UTR结构,我们推测p13基因为一早晚期基因,并主要在早期发挥作用。以Ha-p13为例,我们证明p13基因启动子从2h.p.i到90h.p.i均有活性。不管在天然Hz-AM1细胞还是在异源Sf9细胞中,Ha-p13基因启动子本身并不是一个强启动子,但在hr4存在时,Ha-p13基因启动子活性在Hz-AM1细胞中提高了20倍,在Sf9细胞中则提高了2000倍。携带hr4增强子的Ha-p13基因启动子活性在天然细胞和异源细胞中存在的巨大差异可能受细胞的某些因子调控。为研究P13蛋白功能,我们以Ha-P13和Ls-P13为例,分别对P13蛋白在天然和非天然宿主昆虫细胞和幼虫中的作用进行了研究。由于携带eGFP的棉铃虫杆状病毒HaSNPV-G本身带有Ha-p13基因,因此我们通过dsRNA-Hap13来沉默Ha-P13的表达。流式细胞仪检测结果发现5μg dsRNA-Hap13可以有效地抑制Ha-P13在Sf9和Hz-AM1细胞中的表达。HaSNPV-G与5μg dsRNA-Hap13共感染发现棉铃虫的死亡率明显下降,而且与dsRNA-Hap13的使用剂量成正相关性。这表明p13在天然系统中是一个杀虫相关基因。另一方面,由于I型核型多角体病毒如AcMNPV中缺少p13基因,因此我们将Ls-p13基因插入AcMNPV基因组构建重组病毒rAc-hr5/IE1-Lsp13-G来检验异源系统中P13的杀虫活性。生物测定实验进一步证明,Ls-P13能够明显提早AcMNPV的杀虫时间。通过杆状病毒Ls-P13或Ha-P13与不携带Ls-p13的AcMNPV共同注射感染幼虫发现,一旦Ls-P13的亮氨酸拉链结构发生突变,Ls-P13的杀虫活性也会随之失去,尽管我们对这种确切机理还没有完全解释清楚,但是P13提早杀虫时间的特性很可能使其成为未来新型生物杀虫剂的侯选基因。为进一步研究P13在异源系统中提早杀虫的机理,我们构建了一系列重组病毒来研究P13对病毒在细胞中增殖的影响。我们发现,P13在Sf9细胞中的表达可以明显抑制多角体的产生,其抑制效率随P13表达时相的提前而加强,即P13的早期表达有更强的抑制作用。当P13的亮氨酸拉链发生突变时,P13对多角体的抑制效率随之丧失。而P13的跨膜区发生缺失时,对多角体的效应与Leu Zipper相反,即抑制效果明显提高。另一方面,P13对出芽病毒(BV)的效应与对多角体的效应不同,我们发现,P13在Sf9细胞中的表达可以明显增加出芽病毒(BV)的产生,当亮氨酸拉链突变时,P13对BV的增殖效率随之丧失;当P13的跨膜区发生缺失时,P13对BV的增殖略有下降。以上结果充分证明P13提前杀虫效果的机制是由于P13改变了BV和多角体之间的动态平衡,主要是由于P13的LZLD(Leucine zipper like domain)减少了多角体产量,增加了BV产量。跨膜区使P13晚期定位在质膜上,与LZLD效果相反的是,当TM缺失后P13的定位向核中转移,对多角体的抑制进一步加强,而对BV增加略有下降。BV的参加则加速了AcMNPV从细胞到细胞的次级感染,所以使注射感染时间大大提早。此外,我们对Ha-P13在天然Hz-AM1细胞和Ls-P13在异源Sf9细胞中的定位进行了研究。得出了一致的结果,我们发现P13不管在天然细胞还是异源细胞中,转染48h后主要定位在细胞质膜上。Ls-P13在异源Sf9细胞中的定位为一动态过程,即早期(12h)定位于细胞核中,随后(24h)从细胞核向细胞质膜移动,最后定位于细胞质膜上。当Ls-P13的I、II跨膜区依次缺失时,Ls-P13的晚期定位逐渐从细胞质膜向细胞核移动。尤其在跨膜区II发生缺失时,绝大部分P13蛋白已脱离了细胞质膜而进入核中。由于多角体在核中装配而BV主要在细胞质膜上装配,而且Ls-P13具有抑制多角体产生和促进BV生成的作用。因此当跨膜区缺失时,Ls-P13的定位从细胞质膜而向核移动,核中的Ls-P13的增加和细胞质膜上Ls-P13的降低很好地解释了为什么在跨膜区缺失后L s-P13对多角体的产生的抑制效果进一步加强而对BV的增强效果稍有下降。另一方面,由于L s-P13早期主要在核中表达,随后从核中向细胞质膜上移动,这也很好地解释了为什么L s-P13对多角体的抑制效果随表达时相的推迟而减弱。更多还原

【Abstract】 p13 gene of Leucania separata multinuclear polyhedrosis virus (Ls-p13) was first described by our laboratory as early as 1995. With the large-scale genome sequencing, more than 20 p13 homologues in baculoviruses have been reported. Interestingly, all the reported p13 genes were specifically present in Group II nucleopolyhedroviruses (NPVs) and granuloviruses (GVs), but not in Group I NPVs. The character of p13 promoter and the possible functions of P13 have been predicted in several articles, but there is no evidence in experiment by far. In this study, we have studied on p13 promoter activity, its phylogenetic tree, its killing activity in homologous and heterologous system and possible mechanism. The work set up the foundation for further study of P13 function and its potential application as an opponent of bio-insecticide.Twenty-two p13 sequences from fourteen kind of baculoviruses were reported to date. Fourteen sequences ( each represents one kind of baculovirus P13) selected from the above bacouloviruses genome and were analyzed by DNASTAR software. All the p13 genes share homology from 42.1%-74.7% in amino acid. Among all the sequences, Ls-p13 share the highest homology with HaSNPV p13 in amino acid (58.7%), while share the lowest homology with PoGV p13 in amino acid (44%). Based on the phylogenetic relationship of amino acid, P13 can be clearly divided into two groups in phylogeny, which is p13 genes of GVs and Group II NPVs, respectively. p13 genes of GVs are closely related (58.2%-74.7% amino acid identity) while those of Group II NPVs appear to be more divergent (44.4%-73% amino acid identity). It can be concluded that GV may form a single group according to their close relationship of p13 gene, while NPV containing p13 gene could be divided into subgroup I (LsNPV,HaNPV and SlNPV) and subgroup II (other group II NPVs).p13 gene contains both GTGTTATA box and CAT/AT box of the early promoter, a TTAAG box of late promoter in its 5’-UTR, a mini cistron following the core sequence of the early promoter, and homologue repeats (hr) which is universe in baculovirus. Generally, there are many hrs in baculovirus and the number of p13 hr is different among different baculoviruses. It has been proved that hrs in baculovirus is the ori of DNA replication and enchancer of transcription. hr at p13 gene 5’-UTR is also important for early gene high-expression. Our work confirmed that p13 transcription was regulated by both early and late promoter. Interestingly, we found that the hr4 enhancer/Ha-p13 promoter not only has no host specificity, but also increases its activity in heterogeneous Sf9 cells nearly 100 times more than in homologous Hz-AM1 cells. We presume that some viral or cellular factors may control the normal low-expression of P13 in host cells.In order to make the work more representative, we choose Ha-P13 from Heliothis armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) and Ls-P13 from LsNPV to study the possible killing activity in homologous and heterologous system, respectively. As Ha-p13 was present in the genome of HaSNPV-G (G represents eGFP) and G was not fused with any viral genes, dsRNA-Hap13 could be used as RNA interference to study the effect of Ha-P13 on HaSNPV-G infection. Our results demonstrated that dsRNA-Hap13 could specifically knockdown Ha-P13 expression in both homologous Hz-AM1 cells and heterologous Sf9 cells. Co-injection 5ug dsRNA-Hap13 with HaSNPV-G in larvae could decrease the killing rate and the efficacy was dsRNA-Hap13 dose dependent. Thus p13 gene was indicated as a killing associated gene. On the other hand, as p13 gene was absent in group I NPVs such example as baculovirus AcMNPV, we inserted Ls-p13 gene into AcMNPV to evaluate its killing activity in heterologous system. Bioassay results clearly showed that Ls-P13 could accelerate the killing rate of AcMNPV, but the effect is abolished when its LZLD is disrupted. Such effects were verified both when the protein Lsp13 was expressed in vivo and when a purified sample of the protein was injected into the larvae. Although the mechanism of P13 is still unknown, its killing property is attractive and will promote the further study of P13 functions and its potential applications in biopesticides.In order to probe the possible mechanism of Ls-P13’s killing activity in heterologous system, we constructed a series of recombinant AcMNPVs to study Ls-P13 efffect on polyhedra and BV yield. Phase-contrast microscope and TEM results indicated that Ls-P13 could decrease the polyhedra yield and the inhibition rate decreased with Ls-P13 expression from early to late phase, while the efficacy was lost when LZLD of Ls-P13 was mutated. However, when both transmenbrane domains were deleted, Ls-P13 suppression on polyhedra was further enhanced. On the other hand, our results indicated that BV increased nearly three times when Ls-P13 was highly expressed at early phase. As long as LZLD was mutated, Ls-P13’s effect on BV yield was also lost. However, its increase on BV was still slightly accelerated after the TM deletions. From above results, we concluded that Ls-P13 decreased the yield of polyhedra while increased the yield of BV. LZLD was one of important regions for this efficacy. TMs affected this efficacy only by changes of Ls-P13 distribution. The lost of balance between polyhedra and BV yield may be one reason of Ls-P13 killing activity in heterologous system.Moreover, we have studied the location of P13 in homologous Hz-AM1 cells and heterologous Sf9 cells. Confocal results showed that P13 was located in the cytoplasm membrane at 48h no matter in homologous Hz-AM1 cells or heterologous Sf9 cells. Ls-P13 localization in heterogeneous Sf9 cells was a dynamic process: most of Ls-P13 distributed in the nuclei at early stage (12h); Ls-P13 was then (24h) transferred from nuclei to cytoplasm; Ls-P13 was finally localized cytoplasm membrane. Ls-P13 distribution was transferred from cytoplasm membrane to nuclei with the deletion of TMs, especially after both TM1 and TM2 deletions. Polyhedra are produced in the nucleus while BVs are budded at the cytoplasm membrane. As Ls-P13 distribution was decreased in cytoplasm membrane but increased in nucleus with TM domains deletion, it is well explained why the effect of Ls-P13 with TM mutation on BV increase became weaker while the repression on polyhedra yield was stronger than that of wide type Ls-P13. On the other hand, Ls-P13 would be early-high expressed and enriched in nucleus when Ls-P13 was droved by hr5/IE1 promoter. However, Ls-P13 would move from nucleus to cytoplasm membrane with its expression phase being postponed. It is also explained why Ls-P13 repression efficacy on polyhredra was decreased with its expression from early phase to late phase.更多还原