中文摘要：本发明涉及一种抗螟虫抗草甘膦转基因水稻KCRC04的构建方法。本发明构建了包含草甘膦抗性基因cp4?epsps和抗螟虫基因cry1C的遗传转化载体，通过农杆菌介导的遗传转化方法、以草甘膦作为筛选剂，成功地将载体的T-DNA区单拷贝整合到水稻基因组中，并培育出抗螟虫、抗草甘膦、农艺性状优良的转基因水稻植株KCRC04，明确了外源T-DNA的插入位置并设计了特异性检测方法。本方法适用于转基因水稻的安全评估和检测，以及由该转化事件获得的转基因水稻为供体而得到的各种转基因水稻新品种。
外文摘要：NOVELTY - T-DNA region nucleotide sequence of KCRC04 transgenic rice comprising fully defined 7898 nucleobases (SEQ ID NO: 1) and/or fully defined 1395 nucleobases (SEQ ID NO: 2) given in specifications, is new.
USE - T-DNA region nucleotide sequence of KCRC04 transgenic rice used for controlling weeds and pests (claimed).
DETAILED DESCRIPTION - INDEPENDENT CLAIMS are included for:
(1) detection of KCRC04 of borer-resistant glyphosate transgenic rice comprising extracting total DNA of transgenic rice to be tested; using DNA of first step as template to perform general polymerase chain reaction (PCR) or real time-PCR amplification using specific primer pairs; and analyzing PCR amplification products or real time-PCR amplification curve; and
(2) contruction of KCRC04 comprising taking synthetic glyphosate and borer resistance genes cp4epsps and cry1C; constructing gene synthesized in first step into same genetic transformation vector pZHZH15003; introducing recombinant vector obtained in second step into rice varieties; identifying transformants of third step by molecular identification, screening T0 transgenic plants with single copy insert and no vector backbone and harvesting T1 generation seeds; after 15th day of germination of T1 generation seed in fourth step, spraying glyphosate, selecting transgenic rice lines with glyphosate resistance and Mendelian inheritance to detect exogenous genotypes, and harvesting T2 generation seeds; planting T2 generation seeds, identifying resistance to rice stem borers at different growth stages, and selecting resistant strains with good resistance to insects; cultivating T3 seeds into plants to evaluate agronomic traits of lines, selecting lines with good agronomic traits to detect nucleotide sequence of T-DNA region, and screening transgenic lines with single copy of T-DNA region; and validating transgenic rice plants using previous detection method and finally obtaining anti-borer resista! nt glyph osate-resistant transgenic rice.
主权项：抗螟虫抗草甘膦转基因水稻KCRC04的T?DNA区核苷酸序列，其特征在于，所述T?DNA区核苷酸序列为如SEQ?ID?NO:1所示、或其互补序列或与其同源性达到90%以上的核苷酸序列；所述T?DNA区核苷酸序列的左边界侧翼序列为如SEQ?ID?NO:2所示、或其互补序列或与其同源性达到90%以上的核苷酸序列。
申请号：CN201510565811.0
专利号：CN106497924-A
公开/公告号：CN106497924A
申请日：2015-09-07
公开/公告日：2017-03-15
申请/专利权人：中国种子集团有限公司
发明/设计人：李晨 ; 聂东明 ; 花龙 ; 李旭 ; 李银龙 ; 张琳 ; 孙出 ; 黄炜 ; 刘博林 ; 马崇烈 ; 章旺根
分类号：C12N15/11 ; C12Q1/68 ; C12N15/84 ; A01H5/00
主分类号：C12N15/11
法律状态：实质审查
点击下载：抗螟虫抗草甘膦转基因水稻KCRC04的构建方法