毛白杨PtoYABBY2和PtoYABBY12的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0122

摘要/Abstract

摘要：

目的探究毛白杨PtoYABBY2和PtoYABBY12在开花和叶片发育过程中的作用，为毛白杨分子设计育种提供有价值的参考基因。方法利用同源克隆法克隆毛白杨PtoYABBY2和PtoYABBY12，并对其进行生物信息学、系统发育、表达模式、亚细胞定位分析，通过拟南芥异源转化探究这2个基因的功能。结果毛白杨PtoYABBY2和PtoYABBY12具有完整的YABBY结构域和C2C2结构域，二者属于YABBY基因家族YAB2亚类的基因成员，蛋白均定位于细胞核中。PtoYABBY2和PtoYABBY12在毛白杨雌雄花芽发育过程中呈现不同的表达规律；在毛白杨根、茎、幼叶和成熟叶片中均有表达，且在成熟叶片中表达量最高。与野生型拟南芥相比，PtoYABBY2和PtoYABBY12的过表达植株均表现出明显的早花表型，但叶片形态、花序结构和花器官形态未发生显著变化。过表达转基因株系中，促进开花基因AtFT、AtSOC1、AtFUL和AtLFY相对表达量高于野生型拟南芥，抑制开花基因AtFLC相对表达量显著低于野生型拟南芥。结论毛白杨PtoYABBY2和PtoYABBY12在开花过程中发挥关键作用，可正向促进植物提前开花。

关键词: 毛白杨, YABBY, 开花时间, 表达模式, 功能研究

Abstract:

Objective To explore the role of PtoYABBY2 and PtoYABBY12 genes in the flowering process and leaf development of Populus tomentosa, providing valuable reference genes for molecular design and breeding of P. tomentosa. Method PtoYABBY2 and PtoYABBY12 were cloned by homologous cloning, and bioinformatics, phylogeny, expression pattern and subcellular localization of them were analyzed. The function of these two genes was investigated by allogeneic transformation of Arabidopsis. Result PtoYABBY2 and PtoYABBY12 had complete YABBY and C2C2 domains, both belong to the YAB2 subclass of the YABBY gene family, and their proteins are located in the nucleus. PtoYABBY2 and PtoYABBY12 showed different expression patterns during the development of male and female flower buds in P. tomentosa. Two genes were expressed in the roots, stems, young leaves and mature leaves of P. tomentosa, with the high expression in mature leaves. Compared with wild-type Arabidopsis, the transgenic plants of PtoYABBY2 and PtoYABBY12 both showed obvious early-flowering phenotypes, but no significant changes in leaf morphology, inflorescence structure and floral organ morphology. In the transgenic lines, the relative expressions of flowering-promoting genes AtFT, AtSOC1, AtFUL and AtLFY were higher than those in wild-type Arabidopsis, while the relative expression of flowering-repressing gene AtFLC was significantly lower than that in wild-type Arabidopsis. Conclusion PtoYABBY2 and PtoYABBY12 play a key role in the flowering process, which can positively promote early flowering of plants.

Key words: Populus tomentosa, YABBY, flowering time, expression pattern, functional research

张超超, 韩开元, 王彤, 陈仲. 毛白杨PtoYABBY2和PtoYABBY12的克隆及功能分析[J]. 生物技术通报, 2025, 41(9): 256-264.

ZHANG Chao-chao, HAN Kai-yuan, WANG Tong, CHEN Zhong. Cloning and Functional Analysis of PtoYABBY2 and PtoYABBY12 in Populus tomentosa[J]. Biotechnology Bulletin, 2025, 41(9): 256-264.

图/表 9

表1 引物序列

Table 1 Primer sequence

引物名称 Primer name	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）	用途 Purpose
PtoYAB2	ATGCATCACATAAACGAAAGGAAA	TCACAGGGAGCCATTGATCTCGTA	扩增PCR
PtoYAB12	ATGTCTCTAGACATTGCTTCTGAACG	TTATGCATCCATGCCGGTG	扩增PCR
PtoYAB2-qPCR	CTTTGGACTGAAGCTGGACA	TCACAGGGAGCCATTGAT	RT-qPCR
PtoYAB12-qPCR	TCCCAGTCTTCTTCCTCGGG	GTTCTTGGCTGCATTGCTGA
PtACTIN	TATCTCCTCTGTCTCCGACT	GCATCATCACCTGCAAAC
AtFT （AT1G65480）	CTTGGCAGGCAAACAGTGTATGCAC	GCCACTCTCCCTCTGACAATTGTAGA
AtFUL （AT5G60910）	TTGCAAGATCACAACAATTCGCTTCT	GAGAGTTTGGTTCCGTCAACGACGAT
AtSOC1 （AT2G45660）	AGCTGCAGAAAACGAGAAGCTCTCTG	GGGCTACTCTCTTCATCACCTCTTCC
AtLFY （AT5G61850）	ACGCCGTCATTTGCTACTCT	CTTTCTCCGTCTCTGCTGCT
AtFLC （AT5G10140）	CTAGCCAGATGGAGAATAATCATCATG	TTAAGGTGGCTAATTAAGTAGTGGGAG
AtACTIN2 （AT3G18780）	AGTGGTCGTACAACCGGTATTGT	GATGGCATGGAGGAAGAGAGAAAC

图1 PtoYAB2和PtoYAB12的克隆

Fig. 1 Cloning of PtoYAB2 and PtoYAB12

图2 不同物种YAB2和YAB12的多序列比对

Fig. 2 Multiple sequence alignment of YAB2 and YAB12 from different species

图3 多物种系统发育树

Fig. 3 Phylogenetic tree of multispecies

图4 PtoYAB2和PtoYAB12的亚细胞定位

Fig. 4 Subcellular localization of PtoYAB2 and PtoYAB12

图5 PtoYAB2和PtoYAB12在毛白杨雌雄花芽和不同组织中的表达规律S1‒S8代表8个关键发育时期（S1成花诱导期、S2花原基形成期、S3器官发生期、S4伸长期、S5孢子形成期、S6和S7休眠期、S8孢子发生期）的花芽；R、S、L、OL分别代表根、茎、幼嫩叶片和成熟叶片；♀表示雌花芽，♂表示雄花芽。不同小写字母表示在0.05水平上差异显著，下同

Fig. 5 Expressions of PtoYAB2 and PtoYAB12 in male and female flower buds and different tissues of P. tomentosaS1‒S8 refer to the flower buds at the eight key developmental stages of flower bud development (S1 flower induction stage, S2 flower primordium formation stage, S3 organogenesis stage, S4 elongation stage, S5 archespore formation stage, S6 and S7 dormancy stage, and S8 archespore genesis stage); R, S, L and OL refer to the root, stem, young leaf and mature leaf respectively. ♀ indicates female flower buds and ♂ indicates male flower buds. Different lowercase letters indicate significant differences at the 0.05 level. The same below

图6 转基因拟南芥鉴定与表型分析A：转基因拟南芥阳性鉴定；B：转基因拟南芥与野生型开花时间差异对比；C：转基因拟南芥与野生型叶片表型对比；D：转基因拟南芥与野生型无限花序和花器官对比。YAB2-6、YAB2-8、YAB2-18为PtoYAB2转基因拟南芥株系，YAB12-7、YAB12-8和YAB12-10为PtoYAB12转基因拟南芥株系，下同。B和C中比例尺均为1 cm，D中比例尺从上至下分别为1 cm和1 mm

Fig. 6 Identification and phenotypic analysis of transgenic A. thalianaA: Positive identification of transgenic Arabidopsis thaliana. B: Comparison of flowering time between transgenic A. thaliana and wild type. C: Comparison of leaf phenotypes between transgenic A. thaliana and wild type. D: Comparison of indeterminate inflorescences and floral organs between transgenic A. thaliana and wild type. YAB2-6, YAB2-8, and YAB2-18 are PtoYAB2 transgenic A. thaliana lines, and YAB12-7, YAB12-8, and YAB12-10 are PtoYAB12 transgenic A. thaliana lines, the same below. The scale of B and C is 1 cm, the scale of D from top to bottom is 1 cm and 1mm

表2 野生型与转基因拟南芥抽薹开花时间

Table 2 Bolting flowering time of wild-type and transgenic A. thaliana

株系 Line	光照条件 Light condition	植株数 Number of plants	开花时间 Time of anthesis （d）
WT （Col）	LD	10	20.33±0.36
YAB2-6	LD	11	16.00±0.25**
YAB2-8	LD	12	16.50±0.36**
YAB2-18	LD	10	16.00±0.37**
YAB12-7	LD	12	16.16±0.37**
YAB12-8	LD	12	16.00±0.30**
YAB12-10	LD	10	15.75±0.25**

图7 转基因拟南芥内源基因表达变化分析

Fig. 7 Analysis of changes in endogenous gene expression in transgenic A. thaliana

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