【作者】 皇甫海燕；

【导师】 官春云；

【作者基本信息】 湖南农业大学 ， 作物学， 2011， 博士

【摘要】 油菜受菌核菌侵染时,菌丝必须首先要穿过渗透油菜的细胞壁,所以要分泌一系列降解细胞壁的水解酶。菌核菌分泌的第一个酶就是多聚半乳糖醛酸酶(PG),它会降解油菜细胞壁的主要成分果胶,所以PG是菌核菌重要的致病物质。而多聚半乳糖醛酸酶抑制蛋白(PGIP)可以与PG特异性结合,从而阻止油菜细胞壁的降解,所以对PGIP的研究是十分有意义的。1、本文从菌核菌侵染油菜的机理入手,根据抗菌核病甘蓝型油菜湘油15在接种菌核菌18h后会诱导pgip2基因表达增加,所以提取接种菌核菌18h湘油15的叶片的总RNA,根据甘蓝型油菜pgip2(登录号为EU142024)设计一对特异性引物,以cDNA为模板,RT-PCR扩增其CDS编码序列,应运生物学信息软件对湘油15pgip2核苷酸、氨基酸序列进行分析,并对其蛋白结构进行预测。发现其CDS序列与NCBI库(EU142024)公布的序列同源性为99.7%,其氨基酸序列与NCBI库公布的序列同源性为99.4%。发现湘油15 pgip2编码区CDS长996bp, PGIP2蛋白含331个氨基酸编码的ORF,分子量为37.1KDa,等电点为8.6。预测N端1～22个氨基酸是信号肽,且这一区域疏水性较强。具有5个N-糖基化位点,N端和C端还各具有4个参与二硫键形成的半胱氨酸残基。对其疏水性预测,发现PGIP2是一典型的疏水蛋白。其二、三级结构显示湘油15PGIP2有11个α-螺旋,12个p-延伸链,24个无规则卷曲,蛋白质凹面结构主要由β-折叠/β-转角区构成,凸面结构主要是α-螺旋。中心LRR结构域由5个串联的亮氨酸重复结构域(1 eucine-rich repeat, LRR)基序组成,是由β折叠和a螺旋通过1oop环连接,形成一个马蹄型分子。从第121个氨基酸出现且具有"xLxxLDLSxNxLTGxIPxxLxxL"序列的共同序列,是与PG结合反应的区域。这将为PGIP2蛋白的生物学功能研究提供一定的理论依据。2、将克隆的pgip2 CDS基因亚克隆到原核表达载体pET-32a (+),成功的构建了重组质粒pET-32a-pgip2。该重组质粒以pET-32a系统的起始密码子开始,包括pET-32a载体N端Trx、6×His-Tag、S-Tag和Pgip2基因的序列,C端以Pgip2基因的终止密码子终止。重组质粒转化E.coli BL21(DE3), IPTG终浓度为分别为0.5mmol/L,0.2mmol/L,温度为37℃、25℃,对其重组质粒分别诱导2h,4h,6h,8h,通过SDS-PAGE检测,在预期分子量52KDa处出现表达条带。对其进行可溶性检测,所表达的蛋白都是以包涵体的形式存在,没有可溶形式蛋白的表达。此结论为以后研究植物的Pgip基因在大肠杆菌中的原核表达提供了宝贵的参考依据。3、采用超声波破碎方法裂解包涵体pET-32a-pgip2细胞,通过低浓度变性剂的洗涤液2mol/LUrea、0.5%TritonX-100、1mmol/LEDTA进行洗涤,得到纯度较高的包涵体。再用高浓度的变性剂8mol/LUrea溶解包涵体。通过Ni2+-NTA树脂进行纯化,融合蛋白N端含6×His-Tag会结合在Ni2+柱上,经过不同浓度的咪唑洗脱,最后得到纯度较高的pET-32a-pgip2融合蛋白。纯化后的目的蛋白经Western bolt检测,结果显示,pET-32a-pgip2融合蛋白均可以与抗-His抗体发生免疫学反应,约52KDa处出现一条特异性的反应条带,更进一步的地说明pET-32a-pgip2融合蛋白在大肠杆菌中得到了正确地表达,且读码框正确。纯化的融合蛋白经过肠激酶酶切,再次经过Ni2+-NTA纯化,收集流出液得到PGIP2目的蛋白。最后通过逐渐降低透析液中尿素浓度的透析复性法对目的蛋白进行复性,得到有生物活性的PGIP2蛋白。4、通过对离体油菜叶片滴加PGIP2蛋白,再接种菌核菌菌丝来证明PGIP2蛋白可抑制菌核菌对油菜的侵染,实验显示在不同的天数里,滴加了PGIP2蛋白的叶片比对照滴加了灭菌水、PBS缓冲液的叶片,产生的病斑面积要小,且对油菜的侵害程度也轻,证明PGIP2蛋白对菌核菌侵染油菜有一定的抗性。DNS法测定PGIP2蛋白对PG的抑制作用,数据表明,随着PGIP2蛋白量的增加,PG降解多聚半乳糖醛酸生成D-半乳糖醛酸的量逐渐减少,充分证明PGIP2蛋白对PG有一定的抑制作用。琼脂糖扩散法测定PGIP2蛋白对PG的抑制作用,实验结果发现,随着PGIP2蛋白量在一定量PG里的增加,以多聚半乳糖醛酸作为底物的平板上,圆孔周围透明圈的面积逐渐变小,证明PGIP2蛋白对菌核菌产生的PG有较强的抑制作用,更进一步说明了,大肠杆菌中融合表达的PGIP2蛋白包涵体经过纯化、酶切、复性后是具有生物活性的。5、将克隆的pgip2基因,再次插入到带有CaMV35S强启动子和NOS终止子的植物表达载体pRI101-AN中,构建pRI101-pgip2超表达载体,通过农杆菌介导法转化甘蓝型油菜98c40中,经Kan抗性筛选,获得约300株左右的抗性苗。以CaMV35S启动子特异性引物,对其进行PCR检测,有34株扩增出422bp的目的条带,说明成功获得了转pgip2基因油菜。对其中4株进行半定量RT-PCR检测,发现其中有2株与非转基因植株对比,Pgip2基因表达量上调,并且对这两株油菜的离体叶片接种菌核菌菌丝进行抗性鉴定,发现转基因和非转基因油菜叶片相比,病斑面积明显比较小,受害程度也轻微。结果表明,将Pgip2基因转入了98c40体内并且能够成功的超表达,并且与非转基因对比有效的提高了抗菌核病能力。更多还原

【Abstract】 The mycelium must pass through the cell wall of rapeseed firstly,When rapeseed was infectied by Sclerotinia sclerotiorum so it secreted a series of hydrolases which degraded the cell wall.the first enzyme was poly-galacturonic acid enzyme (PG) which secreted by Sclerotinia sclerotiorum.It can degraded the pectin of rapeseed cell wall,so PG was an important pathogenic substances of Sclerotinia sclerotiorum.Poly-galacturonic acid-inhibiting protein (PGIP) can react with PG spectificity,which preventing rapeseed cell wall to degrade.So the research on PGIP was very significant.1、In this paper, from the mechanism of rapeseed which infected by Sclerotinia sclerotiorum to start research.according to the expression of pgip2 increased after the resistant Sclerotinia sclerotiorum Brassica napus Xiangyou 15 inoculated Sclerotinia sclerotiorum 18 hours.So total RNA was extrated from leaves of rapeseed Xiangyoul5 which were induced with Sclerotinia sclertiorum 18 hours.CDS was amplified from cDNA by reverse transcription PCR (RT-PCR) with a pair of specific primers based on the Brassica napus pgip2 (accession No.EU 142024).Ribonucleotide and amino acid sequence of Xiangyou 15 PGIP were anaylsed. the protein structure were predicted by biological information software.The results showed a homology of the nucleotide and amino acid sequences were 99.7% and 99.4% respectively,which reported NCBI bank.CDS of Xiangyoul5 pgip2 code area was 101 lbp. PGIP2 protein encoding 331 amino acid open reading frame with a molecular mass of 37.1 kDa and isoelectric point of 8.6.1～22 amino acid was predict to the N-terminal signal peptide with stronger hydrophobic region. The deduced amino acid sequence contained 5 potential N-glycosylation sites and contained 4 cysteine residues on N-terminal and C-termianl respectively, which involved in constituting the disulfide linkages.PGIP2 was a typical hydrophobic protein by the hydrophobicity prediction.Secondary and tertiary structure displayed it contained 11α-helices,12β-extended and 24 random coils.The concave structure of protein was composed of P-sheets/β-turns,and convex structure was composed ofα-helices,the center LRR structural domain was composed of 5 tandemly LRRs motifs, LRR consisted of aβ-strands and a-helices connected by loops and forming a horseshone-shaped molecule with the common sequence "xLxxLDLSxNxLTGxIPxxLxxL" form the 121st amino acid,it reacted with PG.It will provided a theoretical basis of PGIP2 protein biological function.2、CDS of PGIP2 was subencoded into the prokaryotic expression vector pET-32a (+) constructing recombinant expression plasmid pET-32a-pgip2 successfully.It started to the initiation codon of pET-32a (+) including the Trx,6×His-Tag, S-Tag of N-terminal pET-32a (+) vector and pgip2 gene sequence.it stopped the stop code of pgip2 gene at C-terminal.The recombinant plasmid was transformed into Escherichia coli BL21 (DE3).The fusion protein pET-32a-pgip was successfully expressed under final concentration 0.2mmolL-1 and 0.5mmolL"1 IPTG at 37℃,25℃iuducing for 2 hours.4 hours,6 hours,8 hours respectively.Expression bands appeared at the place where expected molecular weight 52KDa by SDS-PAGE analysis.It mainly appeared as inclusion bodies,not appeared as soluble protein by testing the solubility of fusion protein. It laid the foundation of the further research about pgip gene of plant prokaryotic expressed in Escherichia Coli.3、Using for ultrasonic crushing methods to broken the cell of pET-32a-pgip2 inclusion bodies and low concentrations of denaturant washing buffer 2mol/LUrea, 0.5% TritonX-100, 1mmol/LEDTA were used to wash it.we obtain high purity inclusion bodies.Then.using the high concentrations of denaturant 8mol/LUrea dissolved inclusion bodies.Using the Ni2+-NTA resin purified it,because the fusion protein containing 6×His-Tag at N-terminal which can combine with the Ni2+-NAT resin, eluting with different concentrations of imidazole, and finally we obtained high purity of the pET-32a-pgip2 fusion protein. the purified protein was analysised by Western bolt,the results show that pET-32a-pgip2 fusion protein can be immune react with anti-His antibody occurring at a specific band at about 52KDa place, further illustrateing that the pET-32a-pgip2 fusion protein was correctly expressed in E.coli,and the reading frame was correct.The purified fusion protein was digested by enterokinase, then was purified by Ni2+-NTA,the effluent liquid was collected to be PGIP2 target protein.Finally,the bioactive PGIP2 protein was obtained by gradually reducing the urea concentration in dialysate and target protein was renatured by the method of dialysis refolding.4、Sclerotinia mycelial was inhibited to infect rapeseed by dropping the PGIP2 protein on the detached leaves and inoculated Sclerotinia mycelial.The research results showed the leaves lesion area was smaller the PGIP2 protein treated leaves than on the sterile water and PBS treated in different days treatment,which demonstrated there was the PGIP2 resistance to Sclerotinia mycelial infected rapeseed. DNS method was used to determine the inhibition effects of PGIP2 on PG.the date showed that with the amount of PGIP2 protein increased,the amount of D-galacturonic acid reduced gradually, which PG degraded poly-galacturonic acid to generate D-galacturonic acid.It adequately proved that PGIP2 protein can inhibit PG.Agarose diffusion method was used to detect the inhibition effects of PGIP2 on PG.The experiment results indicated that with the amount of PGIP2 protein increased in a certain amount of PG, the area of the transparent zone around the plate with poly-galacturonic acid as a substrate lessened gradually.It proved that PGIP2 protein can inhibit PG strongly which Sclerotinia secreted.Further demonstrated PGIP2 fusion protein which was expressed in Escherichia Coil had bioactive after purification,enzymes digestion and renaturation.5、The cloned pgip2 inserted into the palnt expression vector pRI01-AN with CaMV35S strong promoter and NOS terminator.constructing the super-expression vector of pRI101-pgip2. then transferred to Brassica napus 98c40 by Agrobacterium-mediated transformation, about 300 resistant seedlings were got by Kan resistance screening.They were detected for PCR with the CaMV35S promoter-specific primers and 34 plants were amplified 422bp of the target band. This results showed that we got the transgenic rapeseed of pgip2.Four of them were detected by semi-quantitative RT-PCR.The results found that pgip2 gene expression increased compared in two plants with the non-transgenic plants.The detached leaves of the two transgenic rapeseeds were inoculated Sclerotinia mycelial for the resistance indentification.We found that the leaves lesion area of transgenic was smaller than non-transgenic rapeseed leaves lesion, and the invasion was also slight.The results suggested that we transfered the pgip2 into 98c40 and over-expressed successfully.They could improve the resistance to Sclerotinia sclerotiorum than non-transgenic.更多还原