小麦PIN基因家族的鉴定及表达分析

doi:10.3864/j.issn.0578-1752.2020.12.001

摘要/Abstract

摘要：

【目的】PIN（puroindoline）是植物所特有的一类蛋白家族,对控制小麦籽粒硬度有重要功能。分析小麦PIN家族成员在全基因组的分布、结构及进化,研究其在不同组织的表达特异性以及在不同硬度种子的表达模式,为阐明小麦PIN基因家族的生物学功能奠定基础。【方法】根据已报道的小麦PIN基因和大麦HIN基因,利用BLASTP和HMM方法,在最新发布的小麦中国春参考序列中鉴定小麦PIN基因家族成员。利用UniProt、URGI、PFAM、CDD、expVIP等数据库,采用Clustal X、MEGA 7.0、ExPASy、MEME、GSDS、TB tools、GraphPad Prism5等软件进行生物信息学分析。采用qRT-PCR方法检测TaPIN基因家族在不同籽粒硬度小麦样品中的表达情况。【结果】共鉴定出19个小麦PIN基因,集中成簇分布于第1、5和7染色体同源群,编码148—327个氨基酸,编码蛋白相对分子量为16.39—37.19 kD,等电点为6.35—9.34。通过结构域和系统发育分析,可将19个TaPIN基因分为A和B两大类。大部分TaPINs基因仅有1个外显子,没有内含子,顺式作用元件分析发现其上游序列包含大量抗逆和种子发育相关的调控元件。转录组分析表明该基因家族在小麦籽粒中相对表达量很高,而在根茎叶等其他组织几乎不表达。实时荧光定量PCR表明,各基因间相对表达量差异显著,TaPIN9和TaPIN10表达量较高。随着小麦籽粒硬度降低,TaPIN9和TaPIN10表达上调,且表达比例增加,而TaPIN16和TaPIN6则呈现相反的趋势。【结论】小麦籽粒硬度的调节以Pina和Pinb为主,该基因家族其他成员也具有相同结构域,推测也具有相似功能,但受表达量低的限制,对籽粒硬度影响较小。从该基因的进化关系看,粗山羊草与小麦亲缘关系最近,其次是燕麦、黑麦和大麦。

关键词: 小麦, 籽粒硬度, PIN基因, 表达分析

Abstract:

【Objective】 The puroindoline (PIN) gene is a family specific to plants and plays an important role in controlling the grain hardness in wheat. In silico identification and expression analysis of PIN family genes in Triticum aestivum on whole genome level lay the foundation for elucidation the biological function of wheat PIN family. 【Method】 The known sequences of wheat PIN proteins and barley hordoindoline (HIN) proteins were used to query the newly released wheat peptides dataset of Chinese spring variety with HMM and BLASTP profiles. UniProt, URGI, PFAM, CDD, expVIP and other databases were used for bioinformatics analysis with Clustal X, MEGA 7.0, ExPASy, MEME, GSDS, TBtools, GraphPad Prism5 and other programs. The expression profiles of TaPIN genes in different wheat harness seeds were validated with quantitative real-time polymerase chain reaction (qRT-PCR). 【Result】 A total of 19 TaPINs were identified in wheat genome, which were clustered in homologous groups of chromosome 1, 5 and 7. These TaPIN proteins contained 148 to 327 amino acids, as their relative molecular weight varied from 16.39 to 37.19 kD and the isoelectric points ranged from 6.35 to 9.34. Phylogenetic and conserved domain analyses showed that the 19 TaPIN proteins were divided into A and B categories, respectively. Most TaPIN genes harbored only one exon, and there were many cis-acting regulatory elements involved in stress responsiveness and seed-specific regulation. RNA-Seq showed that this gene family expressed largely in wheat grain and hardly expressed in other tissues. The qRT-PCR results indicated that the relative expression level among TaPIN genes were significantly changed. TaPIN9 and TaPIN10 were highly expressed. The expression levels of TaPIN9 and TaPIN10, as well as its expression ratio, were up-regulated with the decrease of wheat grain hardness, while TaPIN16 and TaPIN6 showed an opposite trend. 【Conclusion】 Pina and Pinb genes were the main factors regulating grain hardness in wheat. It is speculated that other members of this gene family have similar functions, but have little influence on grain hardness because of low expression level. According to the evolutionary relationship of this gene, Aegilops tauschii is most closely related to wheat, followed by oats, rye and barley.

Key words: Triticum aestivum, grain hardness, PIN gene, gene expression

刘培勋,万洪深,郑建敏,罗江陶,蒲宗君. 小麦PIN基因家族的鉴定及表达分析[J]. 中国农业科学, 2020, 53(12): 2321-2330.

LIU PeiXun,WAN HongShen,ZHENG JianMin,LUO JiangTao,PU ZongJun. Genome-Wide Identification and Expression Analysis of PIN Genes Family in Wheat[J]. Scientia Agricultura Sinica, 2020, 53(12): 2321-2330.

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基因名称 Gene name	正向引物序列 Forward primer sequence (5′-3′)	反向引物序列 Revers primer sequence (5′-3′)
TaPIN6	TATGCCGCTCTCTTGGGT	GATCGCCTTGGATTGATG
TaPIN7	AGCTATGCAAGCTCCCAC	CACAACTTCTCTTCCCCC
TaPIN8	TATGCCGCTCTCTTGGTT	GATCGCCTTGGATTGATG
TaPIN9	AGCTCCTTGGGGAGTGTT	CAGGTTCTTGGCTTCTTG
TaPIN14	GGCGGTGAAGGGTTTTTC	GCTATCGGGCGTAGTTGC
TaPIN15	AAGGATTATGTGATGGAG	GCTGGTAACACTGGTCTA
TaPIN16	AAAGAAGTGCCGATGTGAGG	GCTGAAAGCCAAAGACGC
TaPIN19	TGTGAACAAGAAGCCCTA	TGCTGAAAACCAAAGATG
TaPIN10	TGAGCATGAGGTTCGGGA	TTGCACTTTGAGGGGAGG
β-Actin	ATGTACCGTGGTGATGTT	CCTGGTGGCTGGTAGTTG