【作者】 杨川毓；

【导师】 张木清；

【作者基本信息】 福建农林大学 ， 作物栽培与耕作学， 2010， 硕士

【摘要】 甘蔗花叶病是一种世界性的病害,目前病毒基因工程在甘蔗抗花叶病育种中得到广泛的应用。农业部甘蔗生理生态与遗传改良实验室于2005年利用基因枪技术成功的将SrMV-P1基因转入受体材料FN95-1702中,获得6个转基因无性系。本研究以上述转SrMV-P1基因为材料,对T3代转基因甘蔗的遗传稳定性、产质量性状进行评价,并研究在病毒胁迫下转基因甘蔗的活性氧代谢和酚类代谢的变化。结果表明:SrMV-P1基因能够有效地提高甘蔗抗花叶病能力。对转基因甘蔗3个世代的田间发病率进行调查,转基因甘蔗的发病率都低于5%,转基因甘蔗田间发病率明显低于受体材料与空载P7,其中TF64表现高抗,三年间均没有发病。对T3代转SrMV-P1基因甘蔗的SrMV-P1基因进行PCR检测,在转基因甘蔗中仍然能检测到SrMV-P1基因,而FN95-1702未能检测到目的基因。说明SrMV-P1基因在转基因甘蔗中能够稳定遗传。对T3代转SrMV-P1基因甘蔗产质量性状进行评价,结果表明:转基因甘蔗的生长速度明显比非转基因甘蔗快,转基因无性系TF37、TF50、TF53、TF63、TF64和TF67的株高显著高于转基因无性系TF50和对照受体材料与空载P7。除转基因无性系TF64外,其他转基因甘蔗的茎径均与受体材料和空载对照无显著差异。6个转基因无性系的产量均比P7高,且高出5.51%～43.30%,其中TF50、TF53、TF63、TF67的产量比FN95-1702高出了8.22%～21.38%。在工艺性状方面上,6个转基因株系的锤度、转光度、蔗汁糖分、视纯度、重力纯度、甘蔗纤维分分别比FN95-1702高出5.00%～24.36%、10.15%～36.52%、9.61%～37.07%、4.65%～9.73%、3.29%～10.25%、3.01%～21.54%和0.37%～26.26%。对转基因甘蔗T1~T3代的产质量性状进行模糊数学隶属函数综合评价可以看出TF53,TF64在三个世代中始终表现优异,FN95-1702和空载P7的表现较差。对转SrMV-P1基因中蔗糖酸性转化酶在甘蔗工艺成熟期前活性较高,有助于甘蔗的营养生长,随着工艺成熟期的到来,蔗糖中性转化酶的活性逐渐上升,有助于甘蔗的糖分积累。FN95-1702与P7由于受到病毒的侵染,甘蔗糖代谢较为混乱。通过对转SrMV-P1基因抗病的生理生化机制研究表明:在四次甘蔗花叶病毒接种过程中,转基因甘蔗TF53、TF64的H2O2、O2-均呈一定规律性变化,并在接种后期其含量处于一个较低的水平;受体材料FN95-1702和P7在整个接种过程中,H2O2、O2-的变化极不规则,并在接种后期其含量维持在一个较高水平。对比接种前后丙二醛含量,发现受体材料FN95-1702和P7在病毒接种后其丙二醛含量比接种前分别提高了58.59%和42.38%,而转基因无性系TF53和TF64的丙二醛含量并无明显提高。对保护酶的研究表明:转基因无性系TF53、TF64在接种病毒后,POD和SOD的活性维持在一个较高水平,有利于清除多余的活性氧,保护叶绿体细胞结构的完整。在酚类代谢研究中得出:接种病毒胁迫后,转基因无性系TF53和TF64的PAL、总酚、类黄酮最后都维持在一个较高的水平,这对甘蔗的抗病性具有积极地意义。更多还原

【Abstract】 Sugarcane mosaic virus is one of the worldwide diseases in sugarcane production.Genetic engineering is used widely for the sugarcane breeding of resistance-disease.SrMV-P1 gene was transformed to sugarcane cv. FN95-1702 by particle bombardment in the Key Lab of Sugarcane Eco-Physiology& Genetic Improvement, Ministry of Agriculture in 2005, and six transgenic sugarcane clones were obtained. The genetic stability, yield and quality of characteristics of the above transgenic sugarcane were evaluated. Active oxygen metabolism and phenolic metabolism were analyzed in transgenic sugarcane inoculated with SCMV virus. The results were showed as followed:Resistance to ScMV was improved effectively in the transgenic sugarcane mediated with SrMV-P1 gene. The SrMV incidence was investigated from T1 to T3 generation. The result was showed that ScMV incidences of all transgenic clones were 5% or less, significantly lower than the receptor cultivars (FN95-1702, CK1) and the transgenic clones without mediated with SrMV-P1 gene (P7, CK2). The trangenic sugarcane clone TF64 was not infected by SrMV virus. In T3 generation, the SrMV-P1 gene was detected in transgenic sugarcane, but not be detected in FN95-1702 and P7, showing that the SrMV-P1 gene could be genetic stability in transgenic sugarcane clones.Yield and quality characteristics in transgenic sugarcane clones were evaluated in T3 generation. The results were showed thatgrowth rate of transgenic sugarcane clones was higher than the control FN95-1702 and P7. plant height of all the transgenic sugarcane except TF50, were higher than FN95-1702 and P7. Stalk diameter in the transgenic sugarcane clones had not significantly difference than those in FN95-1702 and P7. Yield of all the transgenic sugarcane increased by 5.51%～43.30% when compared with P7,and yield of TF50, TF53, TF63, TF67 increased by 8.22%～21.38% when compared with FN95-1702. According to the quality of characteristics in transgenic sugarcane clones, the results indicated that Brix of transgenic sugarcane increased by 5.00%～24.36%, pol by 10.15%～36.52%, sugar content in sugarcane juice by 9.61%～37.07%, apparent purity by 4.65%～9.73%, gravity purity by 3.29%～10.25%, sugarcane fiber by 0.37%～26.26%, Tonnage sugar content (TSC) by 0.37%～26.26%. The results from the fuzzy comprehensive analysis showed that transgenic sugarcane clones had great performance in T1-T3 generation, as compared with FN95-1702 And P7.Acid invertase activity in transgenic sugarcane was higher before sugarcane technical maturing stage with comparison to non-transgenic sugarcane, which was helpful to nutritional growth. As technical maturing stage coming, neutral invertase activity in transgenic sugarcane increased, which was benefit to sucrose accumulation. The FN95-1702 and P7 were infected by SrMV, so the glucose metabolism was confused.The studies on physiological and biochemical basis on virus resistance of transgenic sugarcane TF53, TF64 showed thatwhen sugarcane was inoculated with SrMV virus for four times,content of H2O2 and O2- in TF53 and TF64 were rhythmic change. The content of H2O2 and O2- had a low level at the end of inoculation. During the whole inoculation process, content of H2O2 and O2- in P7 and FN95-1702 were change irregularly, then at the end of inoculation process, the content of H2O2 and O2- had a high level. Comparison of MDA in transgenic sugarcane pre- and post inoculation, the result showed that after SrMV inoculation, the content of MDA in FN95-1702 and P7 increased by 58.59% and 42.38%. MDA content in transgenic sugarcane TF53 and TF64 had not significantly increased after inoculation. The research on protective enzyme system indicated thatthe activities of POD and SOD in transgenic sugarcane were maintained in high level, as compared to non-transgenic sugarcane P7 and FN95-1702, which was used to scavenger reactive oxygen species, to protect cell integrity. Vaiations of phenolic metabolism in transgenic sugarcane showed that:the content of PAL,total phenols,flavonoid in transgenic sugarcane TF53 and TF64 were maintained in high level after SrMV-virus inoculation, , as compared to non-transgenic sugarcane P7 and FN95-1702.更多还原