林业科学 ›› 2021, Vol. 57 ›› Issue (5): 77-92.doi: 10.11707/j.1001-7488.20210508
苗雅慧,鞠丹,梁珂豪,王爱斌,刘峻玲,张凌云*
收稿日期:
2020-06-22
出版日期:
2021-07-25
发布日期:
2021-07-09
通讯作者:
张凌云
基金资助:
Yahui Miao,Dan Ju,Kehao Liang,Aibin Wang,Junling Liu,Lingyun Zhang*
Received:
2020-06-22
Online:
2021-07-25
Published:
2021-07-09
Contact:
Lingyun Zhang
摘要:
目的: NUCLEAR FACTOR Y(NF-Y)转录因子在真核生物中广泛存在,通常由NF-YA、NF-YB和NF-YC 3个亚基形成异源三聚体,来结合下游靶基因启动子中的CCAAT顺式作用元件,进而调控植物的生长发育进程,并参与植物非生物逆境胁迫过程。本研究对青杄中PwNF-YB8基因的表达特性及功能进行分析,揭示其参与的生理过程及对非生物逆境胁迫的响应。方法: 根据实验室前期EST测序及RNA-seq转录组数据分析结果获得青杄NF-Ys家族基因序列,克隆得到PwNF-YB8的cDNA序列,并对其编码蛋白进行序列特征和进化树分析。采用qRT-PCR分析PwNF-YB8在不同组织和花粉萌发过程中的表达特性,以及高温、盐胁迫、甘露醇、ABA处理后的表达变化。亚细胞定位揭示PwNF-YB8在细胞中发挥功能的场所。通过酵母双杂交试验、双分子荧光互补试验分别检测PwNF-YB8的转录激活活性以及与PwHAP5的互作情况。农杆菌介导花序侵染法转化野生型拟南芥(WT),获得纯合的PwNF-YB8过表达株系。利用CRISPR/Cas 9技术获得其同源基因AtNF-YB8的突变体。甘露醇和盐处理后测定野生型(WT)、空载体(VC)、突变体株系(nfyb8-cas9#1/12)和过表达株系(L4、L5)的萌发率、幼苗根长,分析比较不同株系对于渗透胁迫和盐胁迫的耐受能力。结果: PwNF-YB8基因的开放阅读框为489 bp,编码162个氨基酸,具有典型的NF-YB保守结构域,并且与白云杉同源基因具有较近的亲缘关系。SqRT-PCR和qRT-PCR结果表明在青杄的根、茎、针叶和花粉中均能检测到PwNF-YB8的表达,其在花粉中的表达量最高。亚细胞定位试验结果显示,PwNF-YB8定位于细胞核、细胞质中。酵母自激活试验显示PwNF-YB8自身无转录激活活性。进一步对青杄幼苗进行高温(42℃)、盐胁迫、甘露醇和脱落酸处理后发现,PwNF-YB8对干旱和盐2种非生物逆境处理明显响应。PwNF-YB8参与了花粉萌发过程,在花粉萌发36 h时表达量最高。酵母双杂交和双分子荧光互补试验证明PwNF-YB8能够与NF-YC亚基中的PwHAP5互作,可能共同参与花粉管发育调控。甘露醇或盐处理下,异源过表达PwNF-YB8基因的拟南芥种子萌发率与野生型差异不显著,但其根长表现出一定的生长优势。结论: 青杄PwNF-YB8能够与PwHAP5互作,参与花粉萌发和花粉管生长过程,并在干旱、盐胁迫响应中发挥作用。
中图分类号:
苗雅慧,鞠丹,梁珂豪,王爱斌,刘峻玲,张凌云. 青杄转录因子基因PwNF-YB8的克隆与功能分析[J]. 林业科学, 2021, 57(5): 77-92.
Yahui Miao,Dan Ju,Kehao Liang,Aibin Wang,Junling Liu,Lingyun Zhang. Cloning and Functional Analysis of Transcription Factor Gene PwNF-YB8 from Picea wilsonii[J]. Scientia Silvae Sinicae, 2021, 57(5): 77-92.
表1
试验所用引物①"
用途 Application | 引物名称 Primer name | 序列 Sequence(5′—3′) | |
实时定量反转录 PCR qRT-PCR | YB8 | F: ATGGCGGAAGCTAGCAGTCCG | R: TCATGACAGATCATTGCCCTG |
EF1-α | F: AACTGGAGAAGGAACCCAAG | R: AACGACCCAATGGAGGATAC | |
YB8-RT-F | F: GGAGGGTGACAATAAGGGATCTTC | R: CTGCATTGTCACCATATGATGCTG | |
Actin2/8 | F: GGTAACATTGTGCTCAGTGGTGG | R: AACGACCTTAATCTTCATGCTGC | |
载体构建 Vector construction | AD-8-F | F: TCCCCCGGGATGGCGGAAGCTAGCAG | R: CGCGGATCCTCATGACAGATCATTGC |
BD-5-F | F: GGAATTCCATATGATGGATCAGCAGCAGCC | R: CGCGGATCCTCAACTGCTGCCATGAG | |
YB8-bifc | ![]() | ![]() | |
HAP5-bifc | ![]() | ![]() | |
YB8-1205 | F: CGGAATTCATGGCGGGAAGCTAGCAGTCCG | R: GGTACCCCTGACAGATCATTGCCCTGC | |
8-SUPER | ![]() | ![]() | |
DT1-BsF-4 | ATATATGGTCTCGATTGTAGCGATTTTCCCATTAGCGTT | ||
DT1-F0-4 | TGTAGCGATTTTCCCATTAGCGTTTTAGAGCTAGAAATAGC | ||
DT2-R0-8 | AACATTAGCAGGAAGACCTCTTCAATCTCTTAGTCGACTCTAC | ||
DT2-BsR-8 | ATTATTGGTCTCGAAACATTAGCAGGAAGACCTCTTC | ||
突变体鉴定 Mutant identification | Crispr 8 | F: TCTGTTGATGGGTTTTGTCTATTTTG | R: GCCTCAACTTTCACTAGTTAAGAAAAAC |
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