Generation and Genetic Analysis of Xa21 Transgenic Rice Line CX8621 Without Selectable Markers

doi:10.16819/j.1001-7216.2016.5130

Abstract

Abstract:

Selectable marker-free and stable inheritance are the basic requirements for safe application of transgenic crops. Following the regulations approved by the National Committee for Transgenic Biosafety, the wide spectrum bacterial blight resistance gene Xa21 was transferred into a widely used restorer line Minghui 86 by using a double right-borders T-DNA vector through Agrobacterium tumefaciens-mediated system. The transgene Xa21 was confirmed by resistance identification, PCR analysis and Southern blot in the progenies. The single-copy homozygous transgenic line CX8621 without selectable marker and vector backbone sequences was obtained from T₃ generation. The transgenic restorer line CX8621 has stable bacterial blight resistance with years of observation and identification. Up to now, CX8621 has self-fertilized to 16th generation with stable inheritance of transgene Xa21 and passed the bio-safety evaluation of small and large scale of field tests (environmental release and productive test).

Key words: transgenic rice, bacterial blight, Xa21, selectable maker-free, stable inheritance

摘要：

无选择标记和转基因稳定遗传是转基因作物安全应用的基本要求。按照我国转基因作物安全评价指南,利用双右边界T-DNA载体系统将广谱抗白叶枯病基因Xa21转入水稻恢复系明恢86。通过抗性鉴定、PCR分析和Southern杂交,跟踪了外源Xa21基因在转基因后代植株的遗传,并在T₃代获得了无选择标记和载体骨架序列的单拷贝抗病纯合系CX8621。多年的观察和鉴定表明转基因恢复系CX8621具有稳定的白叶枯病抗性。目前CX8621已经稳定遗传至T₁₆,并已通过小规模的田间试验(环境释放)和大规模的田间试验(生产性试验)的安全评价。

关键词: 转基因水稻, 白叶枯病, Xa21, 无选择标记, 稳定遗传

CLC Number:

Q755
S11.032

Zhi-hui XIA, Peng-cheng LIU, Li-fen GAO, Dong-feng LIU, Li JIANG, Guang-huai JIANG, Le-qun GUO, Wen-xue ZHAI. Generation and Genetic Analysis of Xa21 Transgenic Rice Line CX8621 Without Selectable Markers[J]. Chinese Journal OF Rice Science, 2016, 30(1): 10-16.

夏志辉, 刘鹏程, 高利芬, 刘栋峰, 姜丽, 江光怀, 郭乐群, 翟文学. 水稻无选择标记Xa21转基因系CX8621的获得与遗传分析[J]. 中国水稻科学, 2016, 30(1): 10-16.

Figures/Tables 9

Fig. 1. Simplified linear structure and enzyme pattern of pBXa21.

Fig. 2. PCR analysis of transgenic positive T0 plants. A, PCR analysis of hpt; B,PCR analysis of Xa21. pMNDRBBin6,Vector without Xa21;pBXa21,Vector with Xa21;MH86,Non-transgenic control Minghui 86.

Fig. 3. Southern blot hybridization analysis of T0 plants. A, Southern blot analysis of Xa21; B, Southern blot analysis of hpt.

Fig. 4. PCR analysis of transgenic T1 plants of CX86 34-3. A, PCR analysis of Xa21; B, PCR analysis of left border; C, PCR analysis of hpt.

Fig. 5. Southern blot hybridization analysis of T1 plants CX86 34-3. A, Southern blot analysis of Xa21; B,Southern blot analysis of hpt. Selectable marker-free transgenic plants are makered as *.

Fig. 6. Southern blot hybridization analysis of T11 plants from CX8621. T-CX8621; N-Minghui 86.

Table 1 Classification standard for rice bacterial blight disease.

病级 Level	抗性反应 Description	抗性评价 Identification
0	病斑长度小于接种叶片剩余长度5.0%;或病斑面积小于 5.0%。	高抗HR
1	病斑长度占接种叶片剩余长度5.1%~12.0%;或病斑面积占叶面积 5.1%~12.0%。	抗R
3	病斑长度占接种叶片剩余长度12.1%~25.0%;或病斑面积占叶面积 12.1%~25.0%。	中抗MR
5	病斑长度占接种叶片剩余长度25.1%~50.0%;或病斑面积占叶面积 25.1%~50.0%。	中感MS
7	病斑长度占接种叶片剩余长度50.1%~75.0%;或病斑面积占叶面积 50.1%~75.0%。	感S
9	病斑长度大于接种叶片剩余长度75.1%;或病斑面积大于叶面积 75.1%。	高感HS

Table 2 Resistance analysis of Minghui 86 and CX8621.

材料 Material	P1	P2	P3	P4	P6	P7	P8	P9	P10
明恢86 Minghui 86	MS	HS	S	S	S	MS	MS	MR	S
CX8621	HR	HR	R	HR	HR	HR	HR	HR	HR

Fig.7. Spectrum identification and resistance index of Minghui 86,CX8621 and IRBB21. 1-10,Xanthomonas oryzae pv. oryzae P1 to P10, respectively; Number below the figure stands for the average disease index(%).

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