【作者】 姚伟；

【导师】 陈如凯； 张木清；

【作者基本信息】 福建农林大学 ， 作物栽培学与耕作学， 2004， 博士

【摘要】 甘蔗花叶病(Sugarcane Mosaic Disease)，是一种严重危害甘蔗生长的病毒性病害。病发时造成甘蔗大量减产。耕作措施、化学药剂等方法均起不到有效的防治作用，国内外研究证明，培育抗病品种是最经济、有效的防治办法。由于甘蔗是高度杂合的无性繁殖作物，遗传背景非常复杂，其抗病性是受微效多基因控制的数量性状，抗病育种群体分离十分广泛，使得通过常规的育种手段很难将抗病性状与优良品质性状结合起来，植物基因工程为抗病毒育种开辟了新的途径。 根据GENBANK已经公布的甘蔗花叶病毒亚组保守氨基酸序列，设计合成两端引物，用RT-PCR方法从高度感病品种Badila叶片中扩增得到了1.8 kb的预期目的片段，回收并纯化目的片段后，将其克隆于pMD-18-T载体上，测序结果表明我们所克隆的核苷酸序列由一个长为1581bp的开放阅读框(ORF)和231 bp长的3′末端非编码区序列(UTR)组成。ORF中包括病毒的完整外壳蛋白(Coat Protein，CP)序列及部分核内含体蛋白b(Nuclear Inclusion b，NIb)基因的C-端序列。与SCMV亚组中其它序列的分析比较，该病毒分离物的CP基因序列与SCMV-D株系同源性最高，达到了96.08％，其氨基酸同源性高达97.44％，其次是SCMV-A，核苷酸序列和氨基酸序列的同源性分别为94.71％和96.81％。 用基因枪(PDS-1000He)轰击法将含有甘蔗花叶病毒外壳蛋白基因的质粒pNUSCP转化Badila的愈伤组织。共轰击了2251个Badila的愈伤组织块，在含有30 mg／L G418的选择培养基上进行抗性筛选后，得到53株再生植株。分子检测后有24株呈阳性，经Southern杂交分析验证，有13个株系中外源基因以多拷贝形式整合到甘蔗基因组中，并可稳定地遗传给亚无性系。 对转基因植株亚无性系13个株系进行了网虫室抗病性试验，摩擦接种表明，除了B4和B31株系各有一株表现出轻微的发病症状外，其它均无发病症状出现，表现高度的病毒抗性，而对照组几乎全部发病。通过对转基因植株亚无性系叶绿素含量、光合参数、活性氧代谢有关的酶活性测定分析，结果表明转基因Badila由于外源CP基因的导入，病毒在植株体内无法正常复制繁殖，从而保护了植株细胞免受因病毒侵染造成的伤害。未转基因Badila接种后病毒迅速繁殖蔓延，受害叶片对活性氧清除能力下降，促使有毒物质活性氧的积累，启动膜脂过氧化，造成膜的损伤。更多还原

【Abstract】 Sugarcane mosaic virus (SCMV) is a very serious virus disease for sugarcane growth that can cause serious yield lost. Farming practice and chemical control is not sufficient enough to control this disease. Most research prove that traditional plant breeding has played an important role in resistant to virus by providing varieties with disease resistance, higher yield and better quality. However, the sugarcane characters and the difficulties during the selection procedure limited the progress in virus resistance breeding. Plant genetic engineering provides a new way for developing virus resistant cultivars.Primers were designed according to the amino acid conservative sequence of SCMV sub-group. The 1.8 kb desired bands were amplified by RT-PCR from the highly infected Badila by SCMV. PCR products wee purified and cloned into pMD 18-T Vector for sequence analysis. The sequencing result of this fragment shows that the sequence comprised 1581bp ORF and 231bp UTR. The ORF encoded a putative protein of full coat protein and partly nuclear inclusion b. The coat protein sequence was most similar to SCMV-D with identities of 96.08% and 97.44% at the nucleotide and acid levels respectively. Secondly it was similar to SCMV-A with homology of 94.71% and 96.81% at the nucleotide and acid levels respectively.In this research, the calluses of Badila were transformed with pNUSCP containing SCMV-CP by bombardment. After selection on the medium containing 30 mg/L G418, 53 regenerated plants were obtained. PCR analysis showed that 24 plants contained the CP gene and NPTII gene. In which 13 plants contained the CP gene confirmed by southern blotting, and could stably inherited to sub-cloning.The 13 sub-cloning of transgenic sugarcane were inoculated with SCMV for virus resistant evaluation in pest-tight-house. The results showed that the transgenic plants represent highly resistant to virus except B4 and B31 clones, and the untransgenic plants were almost infected by SCMV. By analyzing for chlorophyll. photosynthesis and enzymes in the metabolize of reactive oxygen species in transgenic and untransgenic plants, the results also showed that the transgenic plants can protect sugarcane cell from SCMV damage, but in the untransgenic plants, the tissue were seriously harmed by the accumulation of harmful reactive oxygen species because of the infection of SCMV virus.更多还原