番茄YABBY2b功能鉴定及下游基因的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0308

摘要/Abstract

摘要：

【目的】 分析番茄SlYABBY2b功能，解析其果实大小调控机制，为丰富果实发育调控基因网络及挖掘我国番茄优质基因资源提供依据。【方法】 以番茄‘Ailsa Craig’为材料，通过CRISPR/Cas9技术编辑SlYABBY2b，构建yabby2b突变体株系，并分析yabby2b突变体表型变化，通过石蜡切片技术统计果实细胞数目，RT-qPCR分析候选基因在yabby2b突变体中的表达变化，并对候选基因进行启动子调控元件分析。【结果】 从T₀代转化苗中鉴定到3株突变体，为嵌合突变或双等位突变，从T₁代中分离到3个纯合突变体株系，且不包含转基因元件。表型观察发现，与野生型相比，yabby2b突变体的植株变矮、果实变小。进一步分析果实发育过程发现，yabby2b突变体果实发育早期的子房变小，子房的细胞数目变少。分析候选基因表达变化发现，SlYABBY2b突变后细胞分裂抑制子SlFW2.2的表达水平上调，而且还发现SlFW2.2启动子上有YABBY转录因子的调控元件。【结论】 揭示了SlYABBY2b参与番茄株高和果实大小调控，可能是通过调控SlFW2.2表达，进而影响果实发育早期的细胞分裂，最终调控果实大小。

关键词: 番茄, SlYABBY2b, 果实大小, 细胞分裂, SlFW2.2

Abstract:

【Objective】 To investigate the function of tomato SlYABBY2b and elucidate the mechanism of fruit size regulation, thus laying the foundation for enhancing the gene networks involved in fruit development and uncovering valuable tomato（Solanum lycopersicum L.）gene resources in China. 【Method】 Using CRISPR/Cas9 technology, yabby2b mutant lines were generated by editing the SlYABBY2b gene in tomato ‘Ailsa Craig’. The phenotypic changes of the yabby2b mutants were analyzed, followed by quantification of fruit cell numbers through paraffin sectioning. Expression changes of candidate genes in the yabby2b mutants were assessed using RT-qPCR, along with the analysis of promoter regulatory elements of these genes. 【Result】 Chimeric or biallelic mutants were identified in the T₀ generation, while three homozygous mutants were isolated in the T₁ generation, devoid of transgenic elements. Phenotypic observations revealed that yabby2b mutant plants demonstrated the reduced height and smaller fruits compared to the wild type. Further investigation into fruit development indicated that the ovary of yabby2b mutants was smaller with decreased ovary cell numbers during early fruit development. Analysis of candidate gene expressions revealed an up-regulation of SlFW2.2, a cell division suppressor, in response to the SlYABBY2b mutation, along with the presence of regulatory elements of YABBY transcription factors on the SlFW2.2 promoter. 【Conclusion】 These findings suggest that SlYABBY2b may play a role in regulating plant height and fruit size in tomatoes by modulating SlFW2.2 gene expression, thereby influencing cell division in early fruit development and ultimately determining fruit size.

Key words: tomato, SlYABBY2b, fruit size, cell division, SlFW2.2

孙美华, 孙慧贤, 田林林, 苗妍秀, 侯雷平, 齐明芳, 李天来. 番茄YABBY2b功能鉴定及下游基因的表达分析[J]. 生物技术通报, 2024, 40(11): 162-168.

SUN Mei-hua, SUN Hui-xian, TIAN Lin-lin, MIAO Yan-xiu, HOU Lei-ping, QI Ming-fang, LI Tian-lai. Functional Identification of YABBY2b Gene and Expression Analysis of Downstream Genes in Tomato[J]. Biotechnology Bulletin, 2024, 40(11): 162-168.

图/表 8

表1 基因表达量分析引物

Table 1 Primers for gene expression analysis

基因Gene	引物序列Primer sequence（5'-3'）
SlFW2.2	F: GCTGGGATTGACAGGATTGC
SlFW2.2	R: GCATACATTTCACCTGGTCATGC
SlKLUH	F: AGTTCACCCTCCTGGTCCAT
SlKLUH	R: ACCGAATGGTGCAAGCCTTA
SlCSR	F: AGCGGCTTCCATTTCACCTAA
SlCSR	R: ATCACCACCACCACTGTCTG

表2 T0代番茄yabby2b突变体

Table 2 Tomato yabby2b mutant of T0 generation

株系Plant	突变类型Mutation type	CRISPR/Cas9 T-DNA
#1	嵌合突变Chimeric mutation	+
#3	双等位突变Biallelic mutation	+
#7	双等位突变Biallelic mutation	+

图1 T1代番茄yabby2b突变体 A：yabby2b突变体基因序列（WT：野生型；#2、#3、#6：yabby2b突变体；PAM：前间区序列邻近基序；-为碱基缺失）；B：PCR检测突变体中的CRISPR/Cas9 T-DNA元件（1-11：T1代突变体植株），下同

Fig. 1 Tomato yabby2b mutant of T1 generation A: Gene sequence in yabby2b mutants（WT: Wild-type. #2, #3, #6: yabby2b mutant plants. PAM: Protospacer adjacent motif. - indicates a base deletion）. B: CRISPR/Cas9 T-DNA was detected by PCR in mutants（1-11: plants of T1 generation）. The same below

图2 番茄yabby2b突变体植株表型

Fig. 2 Plant phenotype of tomato yabby2b mutant * P<0.05, **P<0.01. The same below

图3 番茄yabby2b突变体果实表型

Fig. 3 Fruit phenotype of tomato yabby2b mutant

图4 番茄yabby2b突变体果实发育过程

Fig. 4 Fruit development of tomato yabby2b mutant

图5 番茄yabby2b突变体子房壁和隔膜细胞数量分析

Fig. 5 Cell number analysis of ovary wall and septum in tomato yabby2b mutant

图6 关键基因表达量分析及启动子调控元件

Fig. 6 Expression analysis of key genes and promoter regulatory elements

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