细胞分裂素合成关键酶IPT基因在小桐子花芽分化和花性别决定中的功能研究

Jatropha curcas was recognized as a potential feedstock plant for biodiesel production due to the presence of 30% to 40% oil content in its seed. However, the seed yield of Jatropha is low which limit it to be used in the biodiesel industry at present. Jatropha is monoecious which has male and female flowers in the same inflorescence. One of the key factors for low yield is Jatropha has very low proportion female flowers which is just about 3% to 7%. Thus, increasing the the total flower number and the proportion of female flowers seems critical for the improvement of Jatropha seed yield. Early we have found exogenous applications of 6-benzyladenine can increase the total flower number and the proportion of female flowers on Jatropha, and then increase the yield of Jatropha seeds significantly. Thereby, we speculate the total flower number and the proportion of female flowers may increase by transgene expression of isopentenyltransferase (JcIPT) gene in Jatropha flower buds. Totally 9 JcIPTs were identified from the Jatropha genomic database and our transcriptome database of flower buds. Expression profiles of the 9 JcIPTs were analyzed in different tissues and different developmental stages of Jatropha by Real-time PCR, and the results showed JcIPT5, JcIPT6 and JcIPT8 expressed at high level in specific tissues while JcIPT2 expressed in whole plant. According to the results above, this study will further screen JcIPT genes which can produce high enzyme activity in transient expression system, and the JcIPT genes will be constructed in the plant expression vector under the control of the JcAPETALA1 (JcAP1) promoter, respectively, and then transform the constructs into Jatropha via Agrobacterium-mediated genetic transformation. The endogenous cytokinin content in flower buds, the total flower number and the proportion of female flowers of transgenic Jatropha will be analyzed. Finally, the genes regulated by cytokinin and related genes of flower bud differentiation will be detected by Real-time PCR in transgenic Jatropha. These results will identify the biological functions of JcIPT in regulating flower bud differentiation and flower sex determination of Jatropha, and also lay the foundation for breeding high yield lines of transgenic Jatropha.

小桐子被认为是最具发展潜力的能源植物之一，但因亩产较低，并未发挥应有的价值，主要原因之一是其雌雄同株异花，雌花比例仅占3%-7%。本课题组研究发现外施细胞分裂素可以提高小桐子小花数目和雌花的比例，并显著增加了种子产量，因此设想通过转基因技术提高小桐子花芽中细胞分裂素合成关键酶基因JcIPT的表达水平，进而提高其产量。我们前期从9个JcIPTs中筛选到3个组织特异表达且较强和1个整株表达的JcIPTs成员，及完成花芽特异性启动子ProJcAP1的克隆与分析。基于以上基础，本项目拟对这4个候选JcIPTs分别进行瞬时表达分析，筛选具有高酶活的JcIPTs，将全长cDNA与ProJcAP1构建表达载体，并转化小桐子；通过对转基因植株花芽的表型、内源细胞分裂素含量以及受细胞分裂素调控基因的表达进行分析，初步阐明JcIPT在小桐子花芽分化和花性别决定中的功能，为进一步培育高产的转基因小桐子奠定基础。

在本项目的支持下，我们以细胞分裂素合成关键酶基因为研究对象，通过转基因的方法明确它们在小桐子花序芽发育中的生物学功能，结果表明：提高小桐子花序分生组织中细胞分裂素的累积，可以有效地促进花序变大和雌花数量的增加，并最终提高了小桐子的产量。主要取得以下进展：1. 将筛选获得的2个活性较强的小桐子细胞分裂素合成关键酶（isopentenyl-transferases, IPT）基因成员--JcIPT5和JcIPT8，以及1个拟南芥的AtIPT4 和1个农杆菌的ipt 基因全长cDNA分别与我们前期筛选的花芽特异性启动子JcAP1和AtAP3融合构建植物表达载体，共构建了8个表达载体。2. 采用本实验室建立的农杆菌介导法转化小桐子，完成了8个载体的小桐子遗传转化，共获得8个载体的652株转基因小桐子植株，最少的载体转基因株有59株，大部分载体的转基因植株达80株以上。3. 完成转基因小桐子植株的小花数目、雌花比例、果实数量和种子产量多个性状的统计分析。目前所有转基因小桐子已经开花结果，其中一部分转基因小桐子已经是第二年开花结果，与野生型小桐子相比，转基因植株均表现为花芽分化显著增多且雌花易发育的表型，初步阐明了细胞分裂素在小桐子花芽分化过程中的调控功能。其中proAtAP3:JcIPT5和proAtAP3:ipt 两个表达载体的27株转基因小桐子表现出了良好的结果性状，这些转基因株的每个果序上的果子数量和种子产量较对照均显著增加，平均每个果序的果实数量达50个，是对照平均每个果序只有12个果实的4.2倍，单个果序的种子重量是对照的2.84倍。这部分材料有望通过进一步培育成为高产的转基因小桐子品系。4. 实验室建立了AtAP3:ipt 转基因小桐子和野生型的花序芽转录组数据库，并从中筛选出了在转基因小桐子花序芽中显著上调和下调表达的功能基因；明确了细胞分裂素促进小桐子花芽分化增多过程中参与的功能基因，为进一步揭示细胞分裂素在调控小桐子花序发育过程中作用的靶基因研究奠定基础。. 这些研究工作为我们今后小桐子的花性状的改良和高产的转基因小桐子的品种培育奠定基础，也为今后的小桐子花芽分化机理研究提供数据支撑。