杨树GABA支路3个基因家族的鉴定和表达分析

【目的】 γ-氨基丁酸(GABA)与植物的生长发育有着密切的联系。结合前期对GABA抑制杨树不定根发育的研究,对GABA支路基因家族的特征和表达模式进行研究,为进一步解释其在不定根发育中的作用奠定基础。【方法】从银白杨(Populus alba)×P. glandulosa ‘84K’(‘84K’杨)基因组中鉴定出GABA支路的PopGAD、PopGABA-T和PopSSADH 3个基因家族成员,并利用生物信息学方法分析其特征,以及与其他物种相关基因家族的亲缘关系;利用qRT-PCR研究外源GABA及其降解抑制剂vigabatrin(VGB)对各基因表达的影响。【结果】①PopGAD、PopGABA-T和PopSSADH 3个基因家族成员数量依次为6、2和2个,启动子序列中主要包含与光、激素和环境等响应相关元件。②系统进化分析表明,PopGAD家族中PopGAD6与家族其他成员亲缘关系较远;PopGABA-T和PopSSADH家族中的两个基因成员亲缘关系较近。③基因表达分析表明,不定根生长过程中GABA支路基因总体上在根中表达量高于茎和叶。外源GABA或VGB处理对PopGAD和PopGABA-T两个基因家族成员在根、茎和叶中的表达量影响程度不同,但对 PopSSADH基因家族的表达则无明显影响。【结论】 GABA支路3个基因家族在‘84K’杨树响应光、激素和环境胁迫等方面具有重要作用。外源GABA和VGB处理对PopGAD和PopGABA-T家族成员的影响较大,并且均在根中表达量最高,表明这两个基因家族在不定根生长过程中发挥着重要调控功能。这为深入解析GABA在树木不定根发生中的作用机制奠定了基础。

Identification of three gene families in the GABA shunt and their expression analysis in poplar

【Objective】Most prokaryotic and eukaryotic organisms contain γ-aminobutyric acid (GABA), a four-carbon non-protein amino acid, which plays a central role in carbon and nitrogen metabolism. The GABA also occurs in mammals, as it is a major inhibitory neurotransmitter; in addition, it is related to growth and development of plants. Based on our recent findings about the role of GABA in inhibiting poplar adventitious root growth, we studied the characteristics of three gene families in the GABA shunt and their expression patterns to provide a basis for the further investigation of GABA’s function in regulating adventitious root development. 【Method】 The members of three gene families, PopGAD, PopGABA-T and PopSSADH were identified from the complete genome sequence of Populus alba × P. glandulosa ‘84K’ (‘84K’ poplar). The gene structures, conserved motifs, physicochemical characters of proteins and promotor sequences were investigated by using bioinformatics as well as examination of the phylogenetic relationships in these gene families between the 11 species. Tissue-cultured poplar seedlings were cultivated for 12 days with different concentrations of GABA or VGB (GABA degradation inhibitor vigabatrin) treatments. The expression pattern of each gene family was analyzed in the different tissues and treatments with exogenous GABA and VGB by qRT-PCR. 【Result】 ① The PopGAD gene family has six members, the exon numbers are 5-7, and the protein molecular weights are 45.8-57.6 ku; the PopGABA-T gene family has two members, both have 19 exons, and their protein molecular weights are 56.5 ku; the PopSSADH gene family also has two members, there are 4 and 20 exons, respectively, and their protein molecular weights are 57.5 and 15.9 ku, respectively. The gene structure analysis revealed that: all gene members have introns and exons, and the same subfamily exhibits a similar exon-intron pattern; all proteins have three motifs; the promoter sequences of the three gene families all contain elements related to light-, hormone-and stress-responses. However, there were some differences. The promoters of PopGAD gene families have elements related to defense, stress response, and flavonoid biosynthesis. However, these elements are absent in PopGABA-T gene families, and PopSSADH gene families only have defense and stress response elements. ② The phylogenetic analysis of the 11 species showed the GAD gene family is divided into 10 clusters. PopGAD1, PopGAD2, PopGAD3 and PopGAD4, PopGAD5 have close relationships. PopGAD6, which has a less close relationship with other gene family members, belongs to cluster IV, whereas other members all belong to cluster I. Both PopGABA-T and PopSSADH gene families have three clusters. PopGABA-T1 and PopGABA-T2 come from a common ancestor belonging to cluster Ⅲ and PopSSADH1, PopSSADH2 have a close relationship and belong to cluster I. ③ The gene expression analysis showed that all genes of three family members in the GABA shunt, except for PopGAD3 and PopSSADH1, display a higher expression in roots than that in stems and leaves during adventitious root growth. Moreover, the expression of PopGAD1 and PopGAD2 is far beyond that of the other four family members whether or not exogenous GABA or VGB were applied. Exogenous GABA treatments up-regulated PopGAD1 and PopGAD2 in stems and leaves under certain concentrations during adventitious root growth but inhibited the expression of PopGABA-T1 in roots and stems. Exogenous VGB treatments significantly promoted PopGAD1 and PopGAD2 expression in leaves, but inhibited them in roots and stems. Exogenous VGB treatments inhibited both PopGABA-T members in roots. The expression of PopSSADH1 was at a very low level under both GABA and VGB treatments. The expression of PopSSADH2 was far beyond PopSSADH1 and was more inhibited in leaves than in the root and stem. 【Conclusion】 In the GABA shunt, six PopGAD members, two PopGABA-T members and two PopSSADH members were identified from ‘84K’ poplar. The bioinformatics analysis showed that three gene families in the GABA shunt have crucial roles in poplar response to light, hormone and environmental stresses. Both GABA and VGB treatments had a greater impact on the expression of both PopGAD and PopGABA-T families, which have higher expression levels in roots, indicating that these two families may play an important role in regulating poplar adventitious root growth, which would lay a foundation for further deciphering the functions of GABA in adventitious root development in trees.