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Brassica juncea Lines with Substituted Chimeric GFP-CENH3 Give Haploid and Aneuploid Progenies on Crossing with Other Lines

机译:用替代的嵌合GFP-CENH3芥菜系与其他品系杂交后产生单倍体和非整倍体后代

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摘要

Haploids and doubled haploids are invaluable for basic genetic studies and in crop improvement. A novel method of haploid induction through genetic engineering of the Centromere Histone Protein gene, CENH3, has been demonstrated in Arabidopsis. The present study was undertaken to develop haploid inducer (HI) lines of Brassica juncea based on the principles elaborated in Arabidopsis. B. juncea was found to carry three copies of CENH3 which generated five different transcripts, of which three transcripts resulted from alternative splicing. Unlike Arabidopsis thaliana where native CENH3 gene was knocked out for constructing HI lines, we used RNAi approach to knockdown the native CENH3 genes. Further, to rescue CENH3 silenced cells, a GFP-CENH3-tailswap construct having N terminal GFP fused to H3.3 tail sequences and synthetic CENH3 histone fold domain sequences was devised. A total 38 transgenic B. juncea plants were regenerated following co-transformation with both silencing and rescue cassettes and transgenics carrying either or both the constructs were obtained. Transgenic status was confirmed through PCR, Southern and qRT-PCR analyses. Co-transformed lines were crossed to untransformed B. juncea or a line expressing only GFP-tailswap. FACS and cytological analyses of progenies revealed partial or complete elimination of B. juncea chromosomes thereby giving rise to aneuploids and haploid. This is the first report in a polyploid crop demonstrating that CENH3 engineering could be used to develop HI lines.
机译:单倍体和双倍单倍体对于基础遗传学研究和作物改良无价。通过拟南芥组蛋白蛋白质基因CENH3的基因工程诱导单倍体的新方法已在拟南芥中得到证实。根据拟南芥中阐述的原理,进行本研究以开发芥菜的单倍体诱导物(HI)系。发现芥菜芽孢杆菌带有三份CENH3拷贝,产生了五个不同的转录本,其中三个转录本是由选择性剪接产生的。与拟南芥敲除天然CENH3基因以构建HI系不同,我们使用RNAi方法敲除天然CENH3基因。此外,为了挽救CENH3沉默的细胞,设计了具有与H3.3尾序列和合成的CENH3组蛋白折叠域序列融合的N末端GFP的GFP-CENH3-tailswap构建体。在与沉默和拯救盒共转化后,总共再生了38个转基因芥菜植株,并且获得了携带任一种或两种构建体的转基因。通过PCR,Southern和qRT-PCR分析证实了转基因状态。使共转化的品系与未转化的芥菜或仅表达GFP-尾巴交换的品系杂交。 FACS和后代的细胞学分析表明,部分或完全消除了芥菜形杆菌的染色体,从而产生了非整倍体和单倍体。这是多倍体作物的第一份报告,证明CENH3工程可用于开发HI品系。

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