An-1调控水稻每穗粒数基因网络的构建

The grain number per panicle is directly associated with rice yield and regulated by the complicated genetic mechanism.The previous study showed that the An-1 encoded a basic helix-loop-helix transcription factor and regulated grain number per panicle in rice.Up-regulation of An-1 could down-regulate expression of LOG,an important cytokinin regulator, which caused reduction of grain number per panicle and yield per plant in transgenic plants, as compared with those in Nipponbare.Conversely, RNAi of An-1 in an indica variety Kasalath generated more grains per panicle than the control plants.Thus,An-1 is antagonistic to cytokinin and a negative regulator of grain number. Based on the preceding studies,NIP-AN1 transgenic plants were generated, in which grain number per panicle was reduced and almost no awn was formed as compared with that in Nipponbare.The An-1 expression was up-regulated and LOG expression was also down-regulated in NIP-AN1 transgenic plants.The transcriptomes of young panicles were then compared between Nipponbare and NIP-AN1 transgenic plants, using high throughput sequencing technique. Among the 133 differentially expressed genes, the genes involved in transcription regulation,phytohormone and signal transduction pathways have been selected and subjected to mRNA in situ hybridization to determine their spatio-temporal expression patterns. The preliminary study identified the Small Panicle (SP) gene, which was expressed in primary and secondary branch primordia and floral primordia.The sp mutant displayed reduced grain number per panicle as compared with wild type plants. According to the above results,the goal of this project are as follows: Firstly,the candidate genes will be determined by whether they are expressed in branch primordia and floral primordia,using mRNA in situ hybridization.Then,the functions of candidate genes in regulation of grain number per panicle will be confirmed by mutant and transgenic analysis.Furthermore, we will study the interactions between the candidate genes and investigate the relationship between candidate genes and An-1. We will also elucidate whether the candidate genes are regulated by cytokinin pathway.Finally,we will construct the gene regulatory network to regulate grain number per panicle,including An-1,cytokinin and candidate genes,and provide applicable information for rice molecular breeding.

每穗粒数是决定水稻产量的重要农艺性状，受到复杂的遗传机制调控。我们已报道An-1是一个调控每穗粒数的重要基因。An-1表达上调会引起细胞分裂素激活酶基因LOG表达下调，导致每穗粒数降低；反之，则每穗粒数上升。因此，An-1是一个每穗粒数的负调节因子，与细胞分裂素作用相拮抗。本研究前期预实验，通过对An-1上调表达的转基因植物及其亲本幼穗的转录组进行比较分析，获得差异表达基因133个。其中名为Small Panicle(SP)基因突变后导致每穗粒数减少，提示SP可能是参与穗粒数控制的An-1的下游基因。基于此，本项目拟通过mRNA原位杂交鉴定差异基因的表达模式，以确定受An-1调控且调节每穗粒数的候选基因，利用遗传学、细胞生物学和分子生物学手段鉴定其功能；进而阐明包括SP在内的候选基因与An-1及细胞分裂素之间的调控关系，从而建立以An-1、细胞分裂素及下游基因组成的每穗粒数的遗传调控网络。

每穗粒数是决定水稻产量的一个重要农艺性状，结合正反向遗传学手段鉴定更多调控每穗粒数的基因，建立每穗粒数调控的基因网络，阐明控制每穗粒数的遗传机制对水稻产量提高及品种改良有着重要意义。我们过去的研究显示An-1 基因是一个每穗粒数的负调节因子。An-1表达上调，每穗粒数降低、单株产量下降；而An-1表达下调，每穗粒数和单株产量上升。在此基础上，我们利用高通量转录组技术和mRNA原位杂交技术筛选调节每穗粒数的候选基因并进行功能研究。. 在本研究中，我们利用mRNA原位杂交技术鉴定了6个在水稻枝梗和小穗发育时期表达的基因， An-2 (SP)，OsAnAR1—OsAnAR5。它们在花序分生组织、一次和二次枝梗原基、小穗原基中有强烈的表达。 我们利用通过图位克隆技术克隆了An-2基因，它编码一个与LOG蛋白相似的赖氨酸脱羧酶，参与细胞分裂素合成。当An-2表达上调，植株内细胞分裂素浓度大幅度升高，一次、二次枝梗数目和每穗粒数减少，单株产量降低。而An-2功能丧失或表达下调，会使每穗粒数增加和单株产量上升。我们对OsAnAR1-OsAnAR5进行了转基因验证(过量表达和CRISPR-Cas9系统敲除)。其中OsAnAR3基因表达上调，可促进一次、二次枝梗数目及每穗粒数的增加，单株产量提高。OsAnAR3被敲除的转基因植株表现每穗粒数减少，单株产量降低。对OsAnAR3-OX和OsAnAR3-CRISPR 植株<1cm小穗的转录组分析，发现多条激素途径受到了调节，包括细胞分裂素、JA和生长素等途径。另外，OsAnAR5-OX植株表现矮化、早抽穗、每穗粒数减少的表型，但该基因CRISPR敲除植株目前未能得到。 . 我们鉴定了3个新的每穗粒数调控基因，An-2、OsAnAR3和 OsAnAR5，并深入研究了An-2和 OsAnAR3。 An-2是一个每穗粒数负调因子，与An-1具有协同作用；而OsAnAR3是一个每穗粒数正调因子，与An-1有拮抗作用。我们的研究结果丰富了水稻每穗粒数调节机制的信息，对于构建水稻每穗粒数的基因网络有极大的帮助；An-2和OsAnAR3对每穗粒数的正反调控机制，在未来水稻的分子育种中具有应用前景。. 关于An-2的研究结果发表在2015年11月的Molecular plant 杂志上。