Functional Identification of YABBY2b Gene and Expression Analysis of Downstream Genes in Tomato

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

Abstract

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

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.

Figures/Tables 8

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

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	+

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

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

Fig. 3 Fruit phenotype of tomato yabby2b mutant

Fig. 4 Fruit development of tomato yabby2b mutant

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

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

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