水稻t - dna插入突变体库的筛选与遗传分析

LI Ai-Hong , ZHANG Ya-Fang , WU Chang-Yin , TANG Wen , WU Ru , DAI Zheng-Yuan , LIU Guang-Qing , ZHANG Hong-Xi , PAN Xue-Biao
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引用次数: 10

摘要

T-DNA标记技术为鉴定植物中新的功能基因提供了一种强有力的策略,成功的关键是发现T-DNA插入突变体并改变表型。在本研究中,我们从两个转化亲本ZH11和ZH15中筛选了通过整合GLL4/VP16-UAS元件的增强子陷阱系统产生的4 416个水稻T1标记系。我们发现许多品系出现了明显的形态突变,包括假纯合突变和分离突变两种类型。突变表型与株高、抽穗日期、叶形、叶色、分蘖数、穗形、小穗数、粒形、类病突变、雄性不育、芒等14种性状相关。其中株高、抽穗期、叶色和雄性不育性的突变频率较高,均在1%以上。株高和叶色的突变频率在不同年份和转化亲本之间变化不显著,但抽穗日期和雄性不育的突变频率变化较大,说明环境对后两个性状的表达有较大影响。通过T1代和T2代的连续共分离分析,我们鉴定出3个t - dna插入突变体,这些突变体具有穗部或小穗畸形,为克隆相关功能基因提供了基础。同时,我们随机选取42个表型突变株系,通过质粒抢救或TAIL-PCR从39个标记株系中获得40个侧翼序列,其中26个为载体骨干序列,14个与水稻基因组序列具有较好的一致性。BlastN结果表明,T-DNA在植物中优先整合到蛋白质编码区。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Screening for and Genetic Analysis on T-DNA-inserted Mutant Pool in Rice

T-DNA tagging technique has provided a powerful strategy for identifying new functional genes in plants, and the key for success is the discovery of T-DNA-inserted mutants with changed phenotype. In this study, we screened 4 416 rice T1 tagged lines generated by enhancer trap system integrated with GLL4/VP16-UAS elements from two transformed parents, ZH11 and ZH15. We found many lines showed obvious morphological mutations, including two types—fake-homozygous mutation and separating mutation. The mutation phenotype was related to 14 kinds of trait such as plant height, heading date, leaf shape, leaf color, tiller number, panicle shape, spikelet number, grain shape, disease-like mutation, male sterility, awn, and so on. Among them, plant height, heading date, leaf color and male sterility had a comparatively high mutation frequency (over 1%). The mutation frequency of plant height and leaf color had no significant change between different years or transformed parents, but the frequency of heading date and male sterility varied greatly, suggesting that environment had a great effect on the expression of latter two traits. By conducting continuously co-segregating analyses in T1 and T2 generation, we identified 3 T-DNA-inserted mutants with malformed panicle or spikelets, which would provide a base for cloning correlative functional genes. At the same time, we selected randomly 42 lines with mutation phenotype and obtained 40 flanking sequences from 39 tagged lines by plasmid rescue or TAIL-PCR, of which, 26 were vector backbone sequence, 14 had good identity to rice genome sequence. The BlastN result showed the T-DNA preferentially integrated into protein-coding region in plants.

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