cry基因工程甘蔗的分子特征与生物学研究

In China, the total of 90% sugar output is sucrose. The tissue borer is the most serious pest in sugarcane cultivation with the character of atacking through the season, which will result in the loss of more than one million tons of sucrose. Due to the lack of resistant germplasm resoure and the cry gene can improvement the resistance efficiently. The copy numbers of exogenetic gene in transgenic plants is an important factor which can affect the genetic stability.. Thus, first this project will focus on the technique，with the characters of accuracy，safty and high efficiency，to estimate the copy number of the exogenetic gene will be established by means of the comparative analysis of the results obtained by the TaqMan real-time qPCR detection through the two methods of the abslute quantitation and double standard curves. After finishing the detection of the exogenous gene of cry1Ac and cry2A in a big transformed population, then the effects of tansformation methods will be known.. Second, the transgene pathway will be taked as the main line. The gene engineering plant population obtained by the same way will be divided into three groups according to the copy numbers of the exogenetic gene. Among them, the low copy number group with 1-2 copies, 3-5 copies represents the moderate level and 6 or more than 6 copies refer to high. The system study on the relation and the effects among the copy numbers, the protein expression, the the insertion locus flanking sequence and the biologic characters will be conducted. And then, the improved transgenic clones with genetic stability will be selected. Besides, the regulation and control ability of two promoters d35S and ubi will be compared and analysed in the research. .The objectives in this project will state the new theory about sugarcane transgenic research, for example, gene-gun bombardment may be produce low copies in sugarcane, establishment the new technique and method. Besides, the breeding material with the characters of sugarcane borer resistance, which is aimed at the control of the borer in sugarcane cultivation and industry. Thus, the research in this project has an important scientific meanings and applied prospects.

甘蔗生产上螟虫最严重，并具全生长季危害特点，年损失蔗糖上百万吨。甘蔗缺乏抗螟虫资源，转cry基因能有效提高抗虫性，外源基因拷贝数是影响其自身表达水平与遗传稳定性的重要因素。项目拟在比较绝对定量和双标准曲线分析结果基础上，建立基于实时定量PCR的外源基因拷贝数鉴定技术，并对转cry1Ac和cry2A 的大群体进行拷贝数鉴定，明确转化途径的拷贝数效应，并以转化途径为主线，据拷贝数分低（1-2）、中（3-5）和高（≥6）三组，系统研究拷贝数、蛋白时空表达、插入位点旁侧序列及其与生物学性状的关系，筛选遗传稳定的转基因系，并研究ubi和d35S的调控能力。项目把产出转基因甘蔗研究有关的新理论、新技术和新方法与获得旨在解决甘蔗产业螟害问题的抗螟虫育种材料相结合，因此，项目研究兼具重要的科学意义和应用前景。

螟虫具全生长季危害甘蔗的特点，年损失蔗糖上百万吨。甘蔗缺乏抗螟虫资源，转cry基因能有效提高抗虫性，外源基因拷贝数是影响其自身表达水平与遗传稳定性的重要因素。项目把产出转基因甘蔗研究有关的新理论、新技术和新方法与获得旨在解决甘蔗产业螟害问题的抗螟虫育种材料相结合，项目研究兼具重要的科学意义和应用前景。.①在系统评价并筛选获得具有低拷贝和甘蔗特异性的甘蔗内标准基因P4H、APRT和CYC的基础上，研究建立了基于绝对定量和相对定量TaqMan实时荧光定量PCR鉴定甘蔗外源基因拷贝数的技术方法。.②基于TaqMan qPCR双标准曲线法，估算了一批转cry1Ac或 cry2A基因甘蔗株系中的外源基因拷贝数：转cry1Ac甘蔗中，1～10 copies/2C占80.26％；转cry2A的甘蔗该拷贝数仅占24％，超过100 copies/2C的高达24％。高拷贝数明显影响甘蔗生长，中等拷贝数有利于抗虫目标性状改良并保持供体其他性状。.③利用ELISA试剂盒检测不同组织部位的Bt蛋白含量，甘蔗叶片最高、蔗茎皮部中等和髓部最低，分蘖期最高、成熟期次之、拔节期最低。特定株系的表达具有稳定性。.④建立了在培养瓶内利用组培苗饲养繁殖条螟技术，经田间试验，获抗螟虫（螟害株率和螟害节率性状的抗螟虫效果超过80.0%）且产量、蔗糖分与供体相当并可稳定遗传的优异转基因系，安全性评价对土壤微生态影响不明显，具有直接作为生产利用或作为杂交亲本利用的价值。.⑤研究了启动子、cry基因拷贝数、Cry蛋白表达及其对转基因系生物学性状的影响， d35S最优，ST-LS1次之，ubi最差，中等拷贝数对抗虫性状的改良并保持优良综合性状是最有利的。.⑥研究了单引物PCR、Genome walking和hi-Tail PCR三种方法，hi-Tail PCR对鉴定和分析甘蔗基因插入位点旁侧序列最有效。.⑦建立了基于环介导等温扩增原理的可视化检测cry1Ac和 bar基因的技术cry1Ac-LAMP和bar-LAMP，特异性强，灵敏性与qPCR相当。.项目实施培养了博、硕士和本科生各2人，2人获国家研究生奖，发表SCI论文 7 篇，总IF=24.342，中文核心1篇；申报发明专利4件，获发明专利授权2件。