【作者】 林毅；

【导师】 林奇英； 谢联辉；

【作者基本信息】 福建农林大学 ， 农药学， 2003， 博士

【摘要】 核糖体失活蛋白（ribosome-inactivating proteins,RIP）具有抗肿瘤和抗病毒等医用价值，以及抗植物病毒和病原真菌等农用价值。它是一种多活性的物质，不同RIP具有各自独特的功能，其潜在活性还在不断发现之中。葫芦科（Cucurbitaceae）植物大多药食同源，其中蕴涵着极其多样的RIP，但多数已知的RIP其基因尚未被克隆，而目前仍无一套快速筛选RIP新基因的方法。葫芦科中的绞股蓝（Gynostemma pentaphyllum）具有抗癌和抗衰老等特殊功效，其中的皂甙和黄酮成分的研究已有大量报道，但其活性蛋白成分未见报道。为此，本研究开展绞股蓝RIP的分离纯化、基因克隆、原核表达以及转基因植物研究。 率先对绞股蓝活性蛋白成分进行研究，从中分离到一种新的核糖体失活蛋白（绞股蓝毒蛋白，Gynostemmin）。它具有很高的抗TMV活性。其分子量约为27kDa。测得其N-端19个氨基酸序列：DINFSLAGADGQTYNTFIA，与其它植物RIP的同源性为10％～73％，与葫芦科RIP的同源性为37％～73％。 根据葫芦科RIP上、下游两段高度保守的氨基酸序列设计简并引物对LY1／LY2，对基因组DNA进行PCR扩增，首次建立了RIP新基因快速筛选体系。从冬瓜和南瓜中分别获得了1个和5个RIP新基因，长度为408bp，编码136aa，与其它葫芦科RIP对应区段的同源性约为35％～85％。 比较发现，19个葫芦科RIP的LY1／LY2区段上完全相同的氨基酸有29个，其中7个在其它科属来源的RIP中也完全保守，包括构成活性中心的3个残基E₁₆₀、R₁₆₃和W₁₉₂。根据LY1／LY2区段同源性构建的系统进化树表明，葫芦科RIP之间的进化关系与其来源植物间的亲源关系大体一致。 通过DNA片段克隆、RACE扩增、cDNA克隆和DNA克隆，从绞股蓝中分离了13个RIP基因，其中10个为cDNA序列，3个是DNA序列（GP609、DNA-750和DNA-8001）。它们之间在碱基水平和氨基酸水平上的同源性都很高，可根据序列上发生缺失的有无及其类型将其分为4组：组1，发生6bp缺失（535 GAAAAC 540，与574～579间的序列完全一样），包括GynostemminⅡ、GP609和CX8405A，编码275aa；组2，缺失87bp（122～208），包括DNA-750和CX820，编码248aa；组3，包括5RACE和DNA-8001，不发生组2的87bp缺失，但因其序列较短，无法确认是否具备组1的6bp缺失；组4，包括GynostemminⅠ、Ⅲ、福建农林大学博士学位论文Iv、v以及EMUzw和RHXJB，既无87 bp缺失也不发生6 bp缺失，编码277aa。其中GP6Og与其它葫芦科犯P的LYI几YZ区段的同源性，碱基水平为47%⁶1%，氨基酸水平为37%⁴9%。SRACE中包含了由23aa组成的信号肤“M又刃妊JC尹乃/A ILLYFGFHIAEC”，同时包含有5’UTR序列，其中含有ko左水序列“AAAAAA，’。 对绞股蓝犯P基因家族及其编码氨基酸进行的比较分析发现，绞股蓝犯P前体成熟过程中可能发生C一末端切除，并因此造成等电点从弱酸性跃迁至强碱性，这种现象以前未见报道。分析结果进一步支持了FJP基因无内含子的观点。5个含有3’uTR的成员中，Gynosteⁱul比另外4个多了两个小的茎环结构和一富含AU的不稳定子元件ARE（AU·rich element），其mRNA的稳定性可能因此受到影响。运用生物信息学软件对绞股蓝班P前体进行碱基组成和密码子偏倚性、氨基酸组成特征、功能位点、亚细胞定位、跨膜结构、核酸结合基序以及空间结构等方面的分析。 分别将GP609和侧出口B转入pGEX一ZT融合表达载体，在大肠杆菌DHS。菌株中实现融合表达。将DNA一8001连接到pET一sa表达载体上，在大肠杆菌BLZI（DE3）菌株中实现天然表达，表达产物对TMV的抑制效果很差，其原因可能是C一末端延伸序列的存在抑制了抗TMV活性的发挥。 分别构建了含有GP609和EMUzw的 pEmu一mcs一植物表达载体，同时构建了含有RH】JB的pKYLx71:3552植物表达载体（zw-pK）。采用三亲交配法将ZW-pK转入土壤农杆菌LBA4404中，用于转化烟草品种K一326，分子鉴定表明绞股蓝RIP基因已经整合到再生烟苗的基因组中并发生了转录。 重点讨论了绞股蓝RIP的应用前景、犯P基因的内含子、以及犯P的C一末端及其延伸序列的修饰和功能等方面的问题。 研究结果为绞股蓝RIP的开发利用奠定了坚实基础，并对探讨其酶学活性、生理功能以及抗病毒机理等RIP研究的难点和热点问题具有一定的指导意义。更多还原

【Abstract】 Many Cucurbitaceae plants were known TO he Chinese medicine herbs and contain ribosome-inactivating proteins (RIP), a group of toxic proteins that have been proposed and demonstrated to play potential use as toxins in the therapy of a variety of human tumors and viruses including HIV. And much interest has been devoted to construct RIP transgenic crops for the protection from pathogen infection. RIP is a multi-activity protein, and different RIPs exhibit unique functions. Thus, a rapid screening method for novel RIP genes is in urgent need. Gynosiemma pentaphyllum, a member of Cucurbitaceae family, is a kind of herb with great value of Chinese medicine and widely manufactured for health care in China. However, there were no reports on active proteins from Gpentaphyllum.A novel RIP named Gynostemmin (27 kDa) was purified from the leaves and stems of Gpentaphyllum. It showed excellent anti-TMV activity. And the N-terminal 19 amino acid residues are ’DINFSLAGADGQTYN TFIA’ (accession number P83206 in SWISS-PROT). which shared 10%~73% identities to other RIPs from plants and 37%~73% to other RJPs from Cucurbitaceae.Six novel RIP gene fragments (408’bp), 1 from Benincasa hispida and 5 from Cucurbit a moschaia. were isolated from Cucurbitaceae by applying a PCR strategy termed ’conserved regions cloning" using a pair of two degenerated primers deduced from the two blocks with strong amino acid conservation locating at the position 66-73 and 195-202 referring to the mature TCS sequence. Compared with the corresponding regions of 19 RIPs from Cucurbitaceae. 29 completely conserved amino acid residues were revealed, and 7 of them were aiso identical within other type I and type II RIPs from other families, including the proposed active amino acid residues (Gluiro. Arg163, and W192 in TCS).The partial coding region of Gynostemmin (609 bp, accession number AY075115 in GenBank) was cloned from the genomic DNA by using a pair of degenerated primers based on its N-terminal amino acid sequence and the highly conserved amino acid residues in the downstream of most RIPs fromCucurbitaceae. 5’RACE extended 33 bp upstream and revealed a signal peptide with 23 amino acid residues: MRVARFCTVVAILLYFGFHiAEC. Five cDNA sequences with 3’UTR were identified by 3 "RACE, the secondary structures of 3’UTR between GynostemminI and GynostemminII- V were remarkably different. And another 4 cDNA and 2 DNA sequences were obtained. They shared high homologies both at nucleotide acid ieve! and amino acid level. According to the 6 bp or 87 bp deletion, the RIP muitigene family from Gpemaphyllum could be divided into four groups. Group 1, including Gynostemmin [I, GP609. and CX8405A. with the 6 bp deletion at the position of 535-540. encoding 275 aa. Group 2. including DNA-750 and CX820, with the 87 bp deletion at the position of 122-208, encoding 248 aa. Group 3, including 5RACE and DNA-8001. without the 87 bp deletion, and the 6 bp deletion were uncertain because of the incomplete sequence. And group 4, including GynostemminI, III~V, EMUZW, and RHXJB. without the deletion of both 87 bp and 6 bp, encoding 277 aa.Bioinformatic analysis were performed to prepro-Gynostemminl on the base composition, codon preference, amino acid composition, pi value, molecular weight, secondary structure, prosite scan, prediction of protein localization sites. blast thtf Arabidopsis thaliana genome, and 3D structure modeling.GST-fusion and native RIP from Gp&nlaphyUum were expressed in E.coli by inserting the GP609 and RHXJB into the pGEX-2T vector, and DNA-8001 into the pET-5a vector, respectively. The native expressed product from E.coli BL21(DE3) strain, however, showed weak activity against TMV.GP609 and ZWEMU were inserted into pEmu-mcs-N, an expression vectorfor monocotyledon. And RHXJB was inserted into pKYLX71:35S2. a suitableexpression vector for dicotyledon, then introduced into Agrobacterium LBA4404 by triparental mating. Transgenic tobacco K-326 infected by the recombinant Agrobacterium was demonstrated by PCR. sequencing, and N更多还原