CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean
CRISPR/Cas9-mediated targeted mutagenesis of GmLHY genes alters plant height and internode length in soybean
复制标题
CRISPR/Cas9介导的GmLHY基因定向诱变改变大豆株高和节间长度
DOI：
10.1186/s12870-019-2145-8
复制
发表时间：
2019-12-18
影响因子：
5.3
通讯作者：
Kong, Fanjiang
中科院分区：
文献类型：
--
作者：
Cheng, Qun;Dong, Lidong;Kong, Fanjiang
背景
大豆（Glycine max）是重要的经济油料和蛋白质作物。株高是影响大豆产量的重要性状，但对大豆株高的分子机制研究较少。CRISPR（成簇的规则间隔短回文重复序列）/Cas9（CRISPR相关系统9）系统是最近开发的用于基因编辑的技术，其已用于编辑作物植物的基因组。
结果
在这里，我们设计了四种gRNA来突变大豆中的四种晚伸长下胚轴（LHY）基因。为了测试gRNA是否可以在转基因大豆植物中适当地发挥作用，我们首先使用发根农杆菌菌株K599在转基因大豆毛状根中测试了CRISPR构建体。一旦确认，我们进行稳定的大豆转化，并获得了19个独立的转基因大豆植株。随后，我们通过自交获得了一个GmLHY的无T1转基因纯合四倍体突变体。对T2代无转基因四重突变体植株的表型进行了观察，结果表明，GmLHY四重突变体表现为株高降低和节间缩短。Gmlhy 1a 1b 2a 2b的内源赤霉酸（GA 3）水平低于野生型（WT），外源GA 3处理可挽救缩短的节间表型。此外，GA代谢途径基因在GmLHY的四重突变体中的相对表达水平与WT相比显著降低。这些结果表明，GmLHY编码MYB转录因子，通过介导GA途径影响大豆的株高。我们还开发了遗传标记，用于识别突变体，用于育种研究。
结论
我们的研究结果表明，CRISPR/Cas9介导的四个GmLHY基因的靶向诱变降低了大豆植株高度，并缩短了出苗后20至35天（DAE）的节间。这些发现为深入了解大豆株高调控网络的机制提供了依据。
Background
Soybean (Glycine max) is an economically important oil and protein crop. Plant height is a key trait that significantly impacts the yield of soybean; however, research on the molecular mechanisms associated with soybean plant height is lacking. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR-associated system 9) system is a recently developed technology for gene editing that has been utilized to edit the genomes of crop plants.
Results
Here, we designed four gRNAs to mutate four LATE ELONGATED HYPOCOTYL(LHY) genes in soybean. In order to test whether the gRNAs could perform properly in transgenic soybean plants, we first tested the CRISPR construct in transgenic soybean hairy roots using Agrobacterium rhizogenes strain K599. Once confirmed, we performed stable soybean transformation and obtained 19 independent transgenic soybean plants. Subsequently, we obtained one T1 transgene-free homozygous quadruple mutant of GmLHY by self-crossing. The phenotypes of the T2-generation transgene-free quadruple mutant plants were observed, and the results showed that the quadruple mutant of GmLHY displayed reduced plant height and shortened internodes. The levels of endogenous gibberellic acid (GA3) in Gmlhy1a1b2a2b was lower than in the wild type (WT), and the shortened internode phenotype could be rescued by treatment with exogenous GA3. In addition, the relative expression levels of GA metabolic pathway genes in the quadruple mutant of GmLHY were significantly decreased in comparison to the WT. These results suggest that GmLHY encodes an MYB transcription factor that affects plant height through mediating the GA pathway in soybean. We also developed genetic markers for identifying mutants for application in breeding studies.
Conclusions
Our results indicate that CRISPR/Cas9-mediated targeted mutagenesis of four GmLHY genes reduces soybean plant height and shortens internodes from 20 to 35 days after emergence (DAE). These findings provide insight into the mechanisms underlying plant height regulatory networks in soybean.