CRISPR-Based Assessment of Gene Specialization in the Gibberellin Metabolic Pathway in Rice
CRISPR-Based Assessment of Gene Specialization in the Gibberellin Metabolic Pathway in Rice
复制标题
基于 CRISPR 的水稻赤霉素代谢途径基因特化评估
DOI：
10.1104/pp.19.00328
复制
发表时间：
2019-08-01
期刊：
影响因子：
7.4
通讯作者：
Huang, Ju
中科院分区：
文献类型：
--
作者：
Chen, Xiao;Tian, Xuejian;Huang, Ju
赤霉素(GA)是植物生长发育的重要天然调节剂。对于GA代谢途径的每一步，在不同的物种中可以发现不同的拷贝数，就像水稻(Oryza Sativa)的四个酶反应步骤中的13个基因一样。一种普遍的观点是，这种基因复制会产生同源基因，以缓冲生物体免受功能丧失(LOF)突变的影响。因此，任何单一同源基因的敲除可能都不会产生什么影响。为了测试这个问题，我们为这些同源物生成了成簇的规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)敲除，并测量了对生长和生殖的影响。令人惊讶的是，我们在这里报告了在每个酶步骤中始终有一个或多个必需基因，LOF突变导致死亡或不育-这表明GA途径没有冗余途径，每个基因家族对GA代谢是必不可少的。在同一基因家族的大多数基因中，我们观察到了株高和不育性的缺陷，这表明重复的成员保留了与GA合成或降解相关的功能。我们鉴定了三个最近多样化的同源物OsKO1、OsKO2和OsKO5的亚功能化以及OsKO_3和OsKO_4中的新功能化。因此，尽管每个步骤的功能是保守的，但在该步骤中复制的进化是多样化的。有趣的是，位于SD1基因座的CRISPR/Cas9品系通常是不育的，而与水稻“绿色革命”相关的自然突变体SD1表现出正常的结实率。总而言之，我们的结果确定了控制GA生产的候选基因，并为植物中四个关键基因家族的进化提供了洞察力。
Gibberellin (GA) functions as an essential natural regulator of growth and development in plants. For each step of the GA metabolic pathway, different copy numbers can be found in different species, as is the case with the 13 genes across four enzymatic steps in rice (Oryza sativa). A common view is that such gene duplication creates homologs that buffer organisms against loss-of-function (LOF) mutations. Therefore, knockouts of any single homolog might be expected to have little effect. To test this question, we generated clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) knockouts for these homologs and measured effects on growth and reproduction. Surprisingly, we report here that there is consistently one or more essential gene at each enzymatic step, for which LOF mutation induces death or sterility-suggesting that the GA pathway does not have a redundancy route and that each gene family is essential for GA metabolism. In most of these genes from the same gene family, we observed defects in plant height and infertility, suggesting that the duplicated members retain functions related to GA synthesis or degradation. We identified both subfunctionalization of the three recently diversified homologs OsKO1, OsKO2, and OsKO5 and neofunctionalization in OsKO3 and OsKO4. Thus, although the function of each step is conserved, the evolution of duplicates in that step is diversified. Interestingly, the CRISPR/Cas9 lines at the SD1 locus were typically sterile, whereas the natural sd1 mutants, related to the "Green Revolution" in rice, show normal setting rates. Collectively, our results identify candidates for control of GA production and provide insight into the evolution of four critical gene families in plants.