细胞分裂素合成基因IPTs在草莓果实发育早期的功能解析

We analyzed the transcriptional data of early fruit development in strawberry and found that the expression level of tRNA-IPTs is steady and the expression level of adenylate-IPTs fluctuates greatly. Adenylate-IPTs are likely to be responsible for the bulk of trans-Zeatin synthesis, wheras tRNA-IPTs are required for cis-zeatin production. cZ has a lower activity than tZ and its level was increased under stress condition. Its role under stress condition was thought to maintain the basic function of plant cell and improve the resistance of plant. The function of tZ was reported to induce cell division and regulate seed development in arabidopsis and rice. Since the function of the two kinds of CKs catalyzed by the two IPTs is different, we proposed that tRNA-IPTs may be housing keeping genes during fruit development and they may not regulate fruit development, whereas adenylate-IPTs may play a great role in the regulation of fruit development. In this study, the diploid woodland strawberry is used. To test our hypothesis, these two kinds of IPTs genes will be knocked out by CRISPR system separately or combinedly. The phenotype of mutants and wild type will be observed, endogenous CKs will be analyzed and RNA-seq will be used to find out important genes affected by CKs and related to the phenotype of mutants. This study is significant theoretically and will lay a foundation for regulating fruit size and improving the yield of strawberry.

我们对草莓果实早期转录组的分析发现，tRNA-IPTs表达比较稳定，而腺苷酸-IPTs表达变化较大。两类IPT分别合成顺式玉米素（cZ）和反式玉米素（tZ）。cZ生物活性很低，tZ活性高。tZ促进细胞分裂，参与调控种子类果实的发育。逆境下tZ浓度下降，cZ浓度稍有上升，维持植物细胞基本的功能，参与植物对逆境的反应。因此我们提出了两个假说：1）tRNA-IPTs及其对应的cZ在草莓果实发育早期可能只有“看家”的功能，不具有调控功能；2）腺苷酸-IPTs及其对应的tZ参与调控草莓果实早期的发育。为验证这两个假说，我们将以森林草莓为研究对象，利用CRISPR技术分别或组合敲除两类IPTs基因，观察突变体果实发育和生长性状的表型，测定细胞分裂素的浓度，并通过转录组测序，找出受不同种类细胞分裂素影响与果实性状改变相关的基因。该研究具有一定的理论创新，同时也为调控果实的大小和提高产量提供理论依据。

草莓（Fragaria vesca）是研究非呼吸跃变型果实生长、发育和成熟的模式植物。我们从二倍体森林草莓基因组中鉴定到7个IPT基因，包括5个ATP/ADP-IPTs和2个tRNA-IPTs。对两类IPT基因进行了鉴定和系统进化分析，发现两类IPT基因具有不同的起源：tRNA-IPT基因非常古老，广泛地分布在所有生物界；而ATP/ADP-IPT基因仅存在于被子植物中。草莓基因表达分析表明两类IPT基因可能主要在其生殖器官的不同组织或发育阶段具有不同的功能，ATP/ADP-IPT基因的扩张与分化可能与花器官的复杂化有关，而tRNA-IPT基因比较保守。我们通过转基因技术改变两类IPT基因的表达量，发现ATP/ADP-IPT 基因表达量改变，显著影响转基因植株的正常生长，生根与长芽，而改变单个tRNA-IPTs基因的表达量，对植株的正常生长没有显著影响。我们通过施用不同细胞分裂素和利用转基因技术改变果实内源的反式玉米素（tZ）浓度，发现对果实发育和品质具有显著的影响。通过转录组测序，找到了受细胞分裂素调控的一些基因，如与细胞分裂以及与细胞壁相关的基因。这些结果支持我们提出的假说：tRNA-IPTs及其对应的cZ在果实发育过程中可能不具有调控功能，而腺苷酸-IPTs及其对应的tZ参与调控果实的发育。该研究结果将为调控果实的大小和提高产量提供理论依据。