棉花色素腺体发育相关基因<i>GhERF105-like</i>的克隆与表达分析

doi:10.7668/hbnxb.20193130

摘要/Abstract

摘要：

为了探析GhERF105-like在棉花色素腺体发育或棉酚代谢中的功能,利用RT-PCR技术克隆了中棉所12的GhERF105-like基因,并进行了生物信息学分析及表达模式分析。结果显示,GhERF105-like编码区为711 bp,编码236个氨基酸。GhERF105-like蛋白分子质量为26.3 ku,理论等电点pI为 7.72;二级结构预测存在22.88%的α-螺旋、13.14%的延伸链、3.81%的β-转角和60.17%的无规则卷曲。GhERF105-like在第91—155位氨基酸处存在1个AP2结构域,预测有2个苏氨酸、1个酪氨酸和1个丝氨酸磷酸化位点位于AP2结构域内。系统进化分析表明,GhERF105-like及其同源蛋白基序组成在不同物种中保守程度较高,GhERF105-like与黄褐棉、达尔文氏棉、雷蒙德氏棉、海岛棉中同源蛋白的基序组成一致。GhERF105-like在有腺体棉中棉所12各个组织中的表达均显著或极显著高于无腺体棉中棉所12显性无腺体;GhERF105-like在不同有腺体棉品种/系叶片中的表达量不同,且均显著高于显性或隐性无腺体棉品种/系。GhERF105-like受ABA、BR诱导显著上调表达,但在MeJA、Eth、NaCl和PEG处理下显著或极显著下调表达。综上所述,GhERF105-like与棉花色素腺体发育密切相关,可能通过ABA、BR、Eth和JA通路调控腺体发育或棉酚代谢。

关键词: 棉花, AP2/ERF转录因子, 序列分析, 表达分析

Abstract:

In order to explore the function of GhERF105-like in the development of pigment glands or gossyol metabolism of cotton,the GhERF105-like gene of Zhongmiansuo 12 was cloned by RT-PCR,and the bioinformatics analysis and expression patterns were analyzed.The results showed that the coding region of GhERF105-like was 711 bp,encoding 236 amino acids.The molecular weight of GhERF105-like was 26.3 ku,and the theoretical pI was 7.72.The predicted secondary structure of GhERF105-like was 22.88% α-helix,13.14% extended strand,3.81% β-turn and 60.17% random coil.GhERF105-like had an AP2 domain at 91—155 aa.Two threonine,one tyrosine and one serine phosphorylation sites were predicted to be located in the AP2 domain.Phylogenetic analysis showed that motifs of GhERF105-like and its homologous proteins were highly conserved in different species.GhERF105-like motifs were consistent with homologous proteins in Gossypium hirsutum,G.mustelinum,G.darwinii,G.raimondii and G.barbadense.The expression of GhERF105-like in each tissue of glanded cotton Zhongmiansuo 12 was significantly or extremely significantly higher than that of dominant glandless Zhongmiansuo 12.The expression levels of GhERF105-like in leaves of different glanded cotton varieties/lines were different,and were all significantly higher than the expressions of dominant or recessive glandless cotton varieties/lines.GhERF105-like was significantly up-regulated by ABA and BR,but significantly or extremely significantly down-regulated by MeJA,Eth,NaCl and PEG treatment.In conclusion,GhERF105-like is closely related to the development of pigment glands in cotton,and may regulate gland development or gossypol metabolism through ABA,BR,Eth and JA pathways.

Key words: Cotton, AP2/ERF transcriptional factor, Sequence analysis, Expression analysis

张曦, 钱玉源, 王广恩, 刘祎, 权月伟, 崔淑芳, 米换房, 李俊兰. 棉花色素腺体发育相关基因GhERF105-like的克隆与表达分析[J]. 华北农学报, 2022, 37(6): 34-41. doi: 10.7668/hbnxb.20193130.

ZHANG Xi, QIAN Yuyuan, WANG Guang'en, LIU Yi, QUAN Yuewei, CUI Shufang, MI Huanfang, LI Junlan. Cloning and Expression Analysis of a Cotton Pigment Gland Formation Related Gene GhERF105-like[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 34-41. doi: 10.7668/hbnxb.20193130.

http://www.hbnxb.net/CN/Y2022/V37/I6/34

图/表 7

图1 GhERF105-like在中棉所12和中棉所12显性无腺体幼胚腺体发育过程中的表达不同大写字母表示1%水平差异显著(P<0.01)。图7同。

Fig.1 Expression of GhERF105-like during gland development in embryos of Zhongmiansuo 12 and dominant glandless Zhongmiansuo 12 Different capital letters are significantly different at 1% level (P<0.01).The same as Fig.7.

图2 GhERF105-like在不同色素腺体类型棉花幼苗叶片中的表达不同小写字母表示5%水平差异显著(P<0.05)。图7同。

Fig.2 Expression of GhERF105-like in leaves of cotton seedlings with different glandular types Different letters are significantly different at 5% level(P<0.05).The same as Fig.7.

图3 GhERF105-like基因PCR产物电泳分析 M.DL2000 DNA Marker;1.GhERF105-like基因扩增。

Fig.3 Electrophoretic analysis of cotton GhERF105-like gene M.DL2000 DNA Marker;1.GhERF105-like PCR amplification.

图4 GhERF105-like蛋白序列灰色底纹标注为AP2结构域;加粗加大字体标注为预测的磷酸化位点。

Fig.4 Amino acid sequence of GhERF105-like The AP2 domain is shaded in gray;The predicted phosphorylation sites are indicated in big and bold.

图5 GhERF105-like蛋白三维结构预测彩色箭头方向为N→C末端。模型维度(

A ˙

): X.40.874;Y.48.799;Z.44.154。

Fig.5 Predicted crystal structural of GhERF105-like Image coloured by rainbow N→C terminus.Model dimensions(

A ˙

): X.40.874;Y.48.799;Z.44.154.

图5 GhERF105-like蛋白三维结构预测彩色箭头方向为N→C末端。模型维度(

A ˙

): X.40.874;Y.48.799;Z.44.154。

Fig.5 Predicted crystal structural of GhERF105-like Image coloured by rainbow N→C terminus.Model dimensions(

A ˙

): X.40.874;Y.48.799;Z.44.154.

图6 GhERF105-like同源蛋白的进化树(A)及保守基序(B)分析

Fig.6 Phylogenetic(A)and motif composition(B)analysis of GhERF105-like homologous proteins

图7 GhERF105-like的时空表达模式 A.GhERF105-like在有腺体棉中棉所12和中棉所12显性无腺体同一组织部位的表达差异;B、C.中棉所12中GhERF105-like受植物激素诱导的表达模式;D.中棉所12中GhERF105-like非生物胁迫下的表达模式。

Fig.7 Expression patterns of GhERF105-like A.Differential expression of GhERF105-like in same tissue of two cotton germplasms;B,C.Expression patterns of GhERF105-like under hormone treatment in Zhongmiansuo 12;D.Expression patterns of GhERF105-like under NaCl or PEG treatment in Zhongmiansuo 12.

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棉花色素腺体发育相关基因GhERF105-like的克隆与表达分析

Cloning and Expression Analysis of a Cotton Pigment Gland Formation Related Gene GhERF105-like

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