【作者】 喻修道；

【导师】 马有志；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 博士

【摘要】 蚜虫是重要的农业害虫,通过吸食植物汁液或传播病毒病可给作物生产造成严重危害。小麦现有种质资源中不仅缺乏有效的抗蚜基因,而且抗性机制不明确,导致抗虫育种进展缓慢。目前,麦蚜防治以喷洒农药为主,但大量使用农药,不仅对人畜有害,而且造成了严重环境污染。通过转基因工程创制小麦抗蚜新种质,是目前急需解决的重要议题。当前应用于小麦抗蚜基因工程的主要有雪花莲凝集素基因(gna)和半夏凝集素基因(pta)等植物凝集素基因,尽管这些基因抗蚜有一定效果,但研究表明gna转基因植物会影响蚜虫天敌的育性,这些基因应用的安全性引起争议。因此,挖掘和利用安全有效的新型抗蚜基因已成为小麦抗蚜基因工程的研究热点。[反]-β-法尼烯【(E)-β-farnesene,EβF】作为大多数蚜虫报警信息素的主要甚至唯一成份,可以使蚜虫产生骚动、从植株上脱落,并吸引蚜虫天敌,从而有效控制蚜虫危害。本研究以欧洲薄荷(Mentha x piperita)、亚洲薄荷(Mentha haplocalyx Briq)、黄花蒿(Artemisia annua)和花旗松(Pseudotsuga menziesii)为实验材料,克隆了其中的EβF合成酶基因并进行了功能分析,而且将MhβFS1基因转入小麦。具体实验结果如下:1.薄荷EβF合成酶基因的克隆及功能分析:(1)利用RT-PCR方法从亚洲薄荷与欧洲薄荷中分离EβF合成酶基因,其中亚洲薄荷有两个不同的cDNA克隆,分别命名为MhβFS1和MhβFS2,MhβFS2与MhβFS1相比,只有一个编码氨基酸残基的差异(第361位V→A),欧洲薄荷中分离的克隆与MhβFS1完全一致;MhβFS1和MhβFS2的基因组序列长度分别为2690 bp和2753 bp,均有6个内含子,且内含子的相位相同(分别为0、1、2、2、0、0)。(2)qRT-PCR实验结果表明MhβFS基因在薄荷根、茎、叶中均有表达,叶片中表达量稍高。(3)获得MhβFS1/MhβFS2融合蛋白,大小为63 kD左右,纯化后制备抗体,抗体效价大于125000。(4)MhβFS1转基因烟草研究:获得转基因植株12株,Southern杂交、qRT-PCR及Western杂交证明了对MhβFS1基因在烟草中的整合和表达。GC-MS结果显示,转基因株系Mh1-1、Mh1-2、Mh1-4和Mh1-6的EβF释放量分别为243、398、644、769 ng/day;驱蚜试验结果显示,转基因烟草植株对蚜虫有一定的驱避作用,与对照相比,Mh1-1、Mh1-2、Mh1-4和Mh1-6转基因株系上的蚜虫数量分别减少7.0%,9. 3%,16.3%和14.0%。2.黄花蒿EβF合成酶基因的克隆及功能分析:(1)从黄花蒿中分离出两个不同的AaβFS cDNA序列,分别命名为AaβFS1和AaβFS2;AaβFS1和AaβFS2有4个编码氨基酸的差异,包括第21位亮氨酸的缺失,50位天冬氨酸取代天冬酰胺,89位亮氨酸取代异亮氨酸,509位甘氨酸取代精氨酸,已将AaβFS1和AaβFS2 cDNA序列提交GenBank,登录号分别为GU294840、GU294841;AaβFS1基因组序列长度为2392 bp,含有6个内含子,相位依次是0,1,2,2,0和0。(2)分别获得AaβFS1和AaβFS2转基因烟草11株和5株。对转基因烟草植株进行了Southern杂交、qRT-PCR和SDS-PAGE等分子检测,确认了AaβFS基因在烟草中的整合与表达。GC-MS结果显示,AaβFS1和AaβFS2转基因烟草植株均能生成EβF,EβF释放量为217~706 ng/day。驱蚜试验显示,转基因烟草植株对蚜虫有驱避作用,与对照相比,Aa1-4-5转基因株系上的蚜虫数量减少了10.0%;蚜虫天敌吸引试验显示转基因烟草植株对大草蛉有吸引作用;在蚜虫与天敌混合试验中,在转基因株系中,Aa1-3-4上蚜虫数量减少16.4%,Aa1-4-5减少23.6%,Aa2-1-2减少10.9%,Aa2-3-6则减少20.0%,说明转基因烟草植株可以通过吸引蚜虫天敌来控制蚜虫虫害。3.花旗松EβF合成酶基因的克隆及功能分析:(1)从花旗松中分离出两个不同的PmβFS cDNA序列,分别命名为PmβFS1和PmβFS2;PmβFS1和PmβFS2 ORF均有2478个核苷酸组成,编码825个氨基酸,PmβFS1和PmβFS2有9个编码氨基酸的差异,相似性为98.9%;PmβFS2基因组序列长度为3846 bp,包含11个内含子。(2)融合蛋白PmβFS1/PmβFS2的大小为94 kD左右,以包涵体的形式存在于沉淀中。(3)经过PCR、qRT-PCR检测,分别获得15株PmβFS1阳性植株和5株PmβFS2阳性植株;GC-MS分析结果显示转基因烟草产生的EβF量过低,可能与PmβFS1和PmβFS2在烟草中的活性不高有关。4. MhβFS1转基因小麦创制:为提高目的基因的表达水平,本研究采用水稻rbcS启动子,并在MhβFS1 5’端引入了水稻rbcS基因的叶绿体转导肽(CTP)、?和kozak序列,3’端引入poly(A)序列,成功构建了适用于小麦基因枪转化的植物表达载体MhβFS1-pG4AB及MhβFS1+CTP-pG4AB;在此基础上进一步构建了适用于小麦农杆菌介导法转化的植物表达载体MhβFS1-pGПUB和MhβFS1+CTP-pGПUB。分别通过基因枪法和农杆菌介导法将其转入扬麦12和科农199,对转基因植株连续进行PCR鉴定和繁殖,已获得T3代转基因种子;选择部分T0株系进行Southern杂交,结果显示MhβFS1基因已整合至小麦基因组,目前正在检测转基因小麦能否释放EβF及其对蚜虫的驱避性。更多还原

【Abstract】 Aphids are major agricultural pests, partly due to their serious physical and economic damage to cultivated plants by sucking nutrients from the phloem or by transmitting plant viruses. However, the plant defence against aphid-resistance among the available wheat germplasm was seldom identified with the mechanism underlying remains unclear. Chemical insecticides provide a simple strategy for wheat aphid control. However, the large-scale application of such chemicals is not in an environmentally friendly way and becoming increasingly unacceptable. Transgenic wheat engineered for enhanced aphids′resistance could be an efficient alternative strategy. Some plant lectins, including Galanthus nivalis agglutinin (gna) and Pinellia ternate agglutinin (pta), have shown to be toxic towards aphids in transgenic wheat. However, it is reported that gna transgenic plants could cause the sterility of predatory ladybird via aphids in its food chain, and biosafety issues related to the application of these genes need to be evaluated. Therefore, other safe and effective genes for aphid control need to be exploited. EβF [(E)-β-farnesene], as the main and sometimes only component of most aphid alarm pheromone, could cause other aphids in the vicinity to become agitated or disperse from their host plant. What is more, EβF can also function as a kairomone to attract the predator of aphids.Here, the EβF synthase genes were isolated from peppermint、sweet wormwood (Artemisia annua) and douglas fir (Pseudotsuga menziesii), respectively. Functional analysis of individual EβF synthase gene was carried out with transgenic tobacco plants obtained. Moreover, one of the isolated EβF synthase genes, MhβFS1, was transferred into wheat for the first time. The results are indicated as following:1. Cloning and functional analysis of the EβF synthase genes from peppermint: (1) MhβFS genes were isolated from Mentha haplocalyx Briq and Mentha x piperita by RT-PCR. Two EβF synthase genes, named MhβFS1 and MhβFS2, were discovered in Mentha haplocalyx Briq. Compared with MhβFS1, there was a substitution of Val to Ala at position 361 for MhβFS2. The sequence of EβF synthase gene from Mentha x piperita was the same as that of MhβFS1. The length of MhβFS1 and MhβFS2 genomic sequence were 2690 and 2753 bp, respectively. Both of them contain six introns with the same intron phase (intron phase of the six introns is 0, 1, 2, 2, 0 and 0, respectively). (2) qRT-PCR confirmed that MhβFS could express in all the three selected organs, the expression level in leaves was somewhat higher than that of the roots and stems. (3) The molecular weight of heterologous recombinant MhβFS1/MhβFS2 was about 63 kD. The recombinant MhβFS1 was purified using HisTrapTM HP before injecting into a rabbit. The titer of the rabbit′s anti-serum was higher than 125 000. (4) Twelve MhβFS1 positive transgenic individuals were identified by PCR, some of them were further analyzed by southern blot, qRT-PCR and western blot. GC-MS showed that transgenic lines emitted EβF at a level of 243 ng per day for Mh1-1, 398 ng per day for Mh1-2, 644 ng per day for Mh1-3 and 769 ng per day for Mh1-4, respectively. Compared with the control plant W38, transgenic lines showed repellence to aphids with the number of aphids on Mh1-1, Mh1-2, Mh1-4, Mh1-6 decreased 7.0%,9. 3%,16.3% and 14.0%, respectively.2. Cloning and functional analysis of the EβF synthase genes from sweet wormwood: (1) Two EβF synthase genes, designed as AaβFS1 and AaβFS2, were isolated from Artemisia annua. The deduced protein of AaβFS1 had four amino acids differing from that of AaβFS2 including a Leu deletion at position 21, a Asp to Asn at position 50, a Leu to Ile at position 89, a Gly to Arg at position 509. The cDNA sequences of AaβFS1 and AaβFS2 were deposited in GenBank under accession number GU294840 and GU294841, respectively. The length of AaβFS1 genomic sequence was 2392 bp, containing six introns with the intron phase of 0, 1, 2, 2, 0 and 0, respectively. (2) Eleven AaβFS1 and five AaβFS2 positive transgenic tobacco plants were identified by PCR. Southern blot, qRT-PCR and SDS-PAGE further confirmed the integration, expression of the transgenes in transgenic lines. According to the results of GC-MS, AaβFS1 and AaβFS2 transgenic tobacco plants could emit EβF at a level of 217~706 ng/day. The number of aphids on transgenic line Aa1-4-5 decreased 10.0% compared with the non-transgenic control W38. Interestingly, transgenic tobacco plants showed special attraction to green lacewings (Chrysoperla carnea). Then four hundred alate aphids and ten lacewings were simultaneously introduced into a setup, twelve hours later, the number of aphids was reduced by approximately 16.4% in transgenic line Aa1-3-4, 23.6% in Aa1-4-5, 10.9% in Aa2-1-2, 20.0% in Aa2-3-6, respectively. Therefore, we would like to presume that, AaβFS1 and AaβFS2 lines could have a pleiotropic effect on aphid behaviors, especially attraction to aphid predators for aphid control.3. Cloning and functional analysis of the EβF synthase genes from Douglas fir: (1) Two different PmβFS cDNA clones, designed as PmβFS1 and PmβFS2, were isolated from Douglas fir. Both the ORF of PmβFS1 and PmβFS2 contained 2478 nucleotide, which encoded 825 amino acids. The deduced protein of PmβFS1 had four amino acids differing from that of PmβFS2 with 98.9% identities. The length of PmβFS2 genomic sequence was 3846 bp, containing eleven introns. (2) The molecular weight of heterologous recombinant PmβFS1/PmβFS2 protein was around 63 kD, existing in the inclusion body. (3) Fifteen PmβFS1 and five PmβFS2 positive transgenic tobacco plants were identified by PCR and qRT-PCR. GC-MS indicated that transgenic tobacco emitted small amounts of EβF, perhaps due to the activity of PmβFS1 and PmβFS2 synthesas were low in transgenic tobacco plants.4. Preliminary researches on MhβFS1 transgenic wheat: The expression vectors both for particle bombardment, i.e. MhβFS1-pG4AB and MhβFS1+CTP-pG4AB, and Agrobacterium-mediated transformation vectors such as MhβFS1-pGПUB and MhβFS1+CTP-pGПUB were constructed with rice rbcS promotor to direct the expression of target genes with chloroplast transit peptide, ?-kozak sequence added to the 5’-end of MhβFS1 and poly(A) sequence to the 3’-end. MhβFS1 was then transformed into wheat cultivar Yangmai 12 and Kenong199 by biolistic and Agrobacterium-mediated methods, respectively, Positive transgenic wheat lines were advanced to T3 generation. Southern blot of some T0 transgenic wheat lines indicated that transgenes were integrated into the genome of transgenic wheat. Further analysis of EβF emission of the transgenic lines and its effect on the bioassay for aphid control is now under way.更多还原