林业科学 ›› 2022, Vol. 58 ›› Issue (9): 117-127.doi: 10.11707/j.1001-7488.20220912
赵凯,樊艳,邹圣强,郇旭辉,杜淑辉,韩有志,王升级*
收稿日期:
2021-12-27
出版日期:
2022-09-25
发布日期:
2023-01-18
通讯作者:
王升级
基金资助:
Kai Zhao,Yan Fan,Shengqiang Zou,Xuhui Huan,Shuhui Du,Youzhi Han,Shengji Wang*
Received:
2021-12-27
Online:
2022-09-25
Published:
2023-01-18
Contact:
Shengji Wang
摘要:
目的: 基于转录组测序分析,在银腺杨84K中鉴定到1个应答盐胁迫的MYB-related基因PagMYBR96,克隆了该基因并对其特征及参与的生物学过程进行分析,为后续基因功能及调控机制研究和林木基因工程育种提供参考。方法: 采用PCR技术从84K杨cDNA中扩增PagMYBR96基因,对其编码的蛋白序列进行理化性质分析。利用生物信息学分析,对该蛋白的保守结构域、进化关系、亲疏水性、跨膜区域、信号肽、磷酸化位点、二级结构进行分析。采用农杆菌介导的烟草叶片瞬时转化试验分析蛋白亚细胞定位情况;通过酵母转化筛选试验分析蛋白的转录激活活性;通过RT-qPCR分析基因的空表达模式;借助基因共表达及功能注释与富集分析解析基因集参与到的生物学过程和代谢通路。结果: 从银腺杨84K中克隆了长度为882 bp的PagMYBR96基因,编码293个氨基酸,分子质量为33 306.33 Da,理论等电点为6.32,含有保守的MYB结构域和特征氨基酸,与长梗柳中的同源蛋白亲缘关系较近,没有跨膜区域,也不存在信号肽,可能是一个亲水性蛋白质。PagMYBR96为核定位蛋白,且在酵母中没有转录激活活性。基因时空表达模式分析结果表明,PagMYBR96在84K杨的根和叶中均可应答盐胁迫,且在胁迫初期上调表达,到达峰值后渐渐恢复。PagMYBR96及与其共表达的518个基因与细胞过程、代谢过程、生物调节、对刺激应答、发育过程等相关,且分别被显著富集到类泛素蛋白结合酶活性、类泛素蛋白转移酶活性、泛素蛋白转移酶活性和泛素结合酶活性等GO term及氨酰-tRNA生物合成、泛素介导的蛋白水解和丙酮酸代谢等通路中。结论: 银腺杨84K PagMYBR96基因可以在不同的组织中应答盐胁迫,并通过不同的生物学过程参与植物的生长发育及胁迫应答。PagMYBR96及其共表达基因的功能与泛素蛋白酶体系统介导的蛋白质降解及丙酮酸代谢等过程相关。
中图分类号:
赵凯,樊艳,邹圣强,郇旭辉,杜淑辉,韩有志,王升级. 银腺杨84K盐胁迫应答基因PagMYBR96的克隆及表达分析[J]. 林业科学, 2022, 58(9): 117-127.
Kai Zhao,Yan Fan,Shengqiang Zou,Xuhui Huan,Shuhui Du,Youzhi Han,Shengji Wang. Cloning and Expression Analysis of PagMYBR 96 Involved in Salt Stress Response in Populus alba × P. glandulosa '84K'[J]. Scientia Silvae Sinicae, 2022, 58(9): 117-127.
表1
本研究使用的引物"
引物名称 Primer name | 引物序列 Primer sequence (5′—3′) |
PagMYBR96-F | GCAGATTCCTGATTCCTTGTGTATG |
PagMYBR96-R | CTAGAAGAAAGCCTCCTGAAACTAG |
PagMYBR96-GFP-F | CTAGTCTAGAATGGAAACTCTGAATCCATC |
PagMYBR96-GFP-R | ACGCGTCGACCATATTGAATTTGAAATCTTG |
PagMYBR96-BD-F | GCCATGGAGGCCGAATTCATGGAAACTCTGAATCCATC |
PagMYBR96-BD-R | TGCGGCCGCTGCAGGTCGCTAATATTGAATTTGAAATC |
PagMYBR96-qPCR-F | CTGGGACGAAGCAAAGGATGGATC |
PagMYBR96-qPCR-R | GTTGATACGAGCCCCATGATGAGC |
Actin-F | ACCCTCCAATCCAGACACTG |
Actin-R | TTGCTGACCGTATGAGCAAG |
表2
与PagMYBR96共表达的转录因子基"
基因ID Gene ID | 转录因子家族 Transcription factor family | 拟南芥中的同源基因 Homologous genes in Arabidopsis |
Potri.010G247200.1 Potri.015G105300.1 Potri.011G129500.1 Potri.014G106300.1 Potri.015G105200.1 Potri.018G141800.1 Potri.014G028200.1 Potri.017G116300.1 Potri.016G053700.1 Potri.013G066700.1 Potri.017G042200.1 Potri.014G007200.1 Potri.011G025000.1 Potri.014G075200.1 Potri.015G034100.1 Potri.011G051600.1 Potri.014G017400.1 Potri.017G086300.1 Potri.014G106800.1 Potri.014G108100.1 Potri.015G034500.1 Potri.013G025000.1 Potri.011G096600.1 Potri.015G138200.1 Potri.013G039100.1 Potri.012G047700.1 Potri.015G039100.1 Potri.014G066700.1 Potri.018G129800.1 | AP2 ARF bHLH bHLH bHLH bHLH bZIP C2H2 C3H Dof GATA GRF HD-ZIP HD-ZIP HD-ZIP HSF LBD MYB MYB_related NAC NF-X1 NF-YC TCP TCP Trihelix WOX WOX YABBY YABBY | AT3G54320.3 AT5G62000.1 AT3G20640.1 AT2G46810.1 AT3G26744.1 AT5G57150.1 AT1G45249.1 AT1G67030.1 AT2G41900.1 AT3G47500.1 AT3G21175.2 AT2G22840.1 AT4G04890.1 AT4G00730.1 AT1G73360.1 AT5G16820.1 AT3G49940.1 AT5G15310.2 AT1G01060.4 AT5G24590.2 AT1G10170.1 AT1G08970.1 AT3G15030.1 AT5G51910.1 AT5G28300.1 AT3G18010.1 AT3G18010.1 AT2G45190.1 AT2G26580.1 |
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