草业学报 ›› 2023, Vol. 32 ›› Issue (1): 178-191.DOI: 10.11686/cyxb2022008
• 研究论文 • 上一篇
刘福1,2(), 陈诚1,2, 张凯旋2, 周美亮2(), 张新全1()
收稿日期:
2022-01-06
修回日期:
2022-02-24
出版日期:
2023-01-20
发布日期:
2022-11-07
通讯作者:
周美亮,张新全
作者简介:
E-mail: zhangxq@sicau.edu.cn基金资助:
Fu LIU1,2(), Cheng CHEN1,2, Kai-xuan ZHANG2, Mei-liang ZHOU2(), Xin-quan ZHANG1()
Received:
2022-01-06
Revised:
2022-02-24
Online:
2023-01-20
Published:
2022-11-07
Contact:
Mei-liang ZHOU,Xin-quan ZHANG
摘要:
干旱是影响植物生长发育的重要环境因素。本研究分析了日本百脉根抗旱相关基因LjbHLH34的耐旱功能,初步解析其响应干旱胁迫的分子机制,以期为百脉根抗旱分子育种提供理论基础。本研究克隆得到的LjbHLH34基因大小为711 bp、编码236个氨基酸,属bHLH转录因子家族成员。系统进化树分析显示,LjbHLH34蛋白与拟南芥bHLH Ⅳ亚家族中AtbHLH34和AtbHLH104亲缘关系较近。实时荧光定量分析表明LjbHLH34在日本百脉根的根中表达量最高,叶中次之,茎中最少,暗示其在日本百脉根多个组织中发挥作用;同时LjbHLH34基因也受聚乙二醇(PEG)和脱落酸(ABA)诱导表达。在酵母中检测发现LjbHLH34具有转录激活活性;亚细胞定位试验表明LjbHLH34蛋白定位于细胞核中。将LjbHLH34基因转入拟南芥获得过表达株系。在200 mmol·L-1甘露醇胁迫下,LjbHLH34转基因拟南芥的根长明显长于野生型。干旱处理后,野生型拟南芥比转基因拟南芥萎蔫程度更加明显,而转基因株系的相对含水量和超氧化物歧化酶(SOD)活性显著高于野生型,丙二醛(MDA)积累显著低于野生型。qRT-PCR检测发现在干旱处理之后,与抗逆相关的基因AtCAT1、AtCAT3和AtRD22在转基因株系中的表达量显著升高。上述结果表明LjbHLH34正调控植物的抗旱性。
刘福, 陈诚, 张凯旋, 周美亮, 张新全. 日本百脉根LjbHLH34基因克隆及耐旱功能鉴定[J]. 草业学报, 2023, 32(1): 178-191.
Fu LIU, Cheng CHEN, Kai-xuan ZHANG, Mei-liang ZHOU, Xin-quan ZHANG. Cloning and identification of drought tolerance function of the LjbHLH34 gene in Lotus japonicus[J]. Acta Prataculturae Sinica, 2023, 32(1): 178-191.
引物名称Primer name | 引物序列Primer sequence (5′-3′) |
---|---|
T-LjbHLH34-F | ATGGTTTCCGCGGAAAACACC |
T-LjbHLH34-R | TTAGGCAGCTGGTGGTCGGAG |
M13-F | TGTAAAACGACGGCCAGT |
pCAMBIA1307-LjbHLH34-F | |
pCAMBIA1307-LjbHLH34-R | |
pCAMBIA1307-F | AGGAAGTTCATTTCATTTGGA |
pAN580-LjbHLH34-F | |
pAN580-LjbHLH34-R | |
PAN580-F | ATGACGCACAATCCCACTATCC |
pGBKT7-LjbHLH34-F | |
pGBKT7-LjbHLH34-R | |
T7-F | TAATACGACTCACTATAGG |
qLj-actin3-F | GTATTGTTGGCCGACCTCGT |
qLj-actin3-R | AGCCTCAGTTAGAAGCACCG |
qLj-bHLH34-F | ATGCAGTTCGAGTGGTGACG |
qLj-bHLH34-R | AGACGGCAAAAATTGCCACA |
qAt-actin7-F | TCCATGAAACAACTTACAACTCCATCA |
qAt-actin7-R | CATCGTACTCACTCTTTGAAATCCACA |
qAtCAT1-F | GAGATCCCCGTGGTTTTGCT |
qAtCAT1-R | TGTGCAAACTCTCTGGGTGG |
qAtCAT3-F | AGCTTCCAGTCAATGCTCCC |
qAtCAT3-R | GTGAGACGTGGCTCCGATAG |
qAtRD22-F | TTCGTCTTCCTCTGATCTGTCTTC |
qAtRD22-R | TTTACTCCGCCTTTACCTACTTGG |
表1 引物序列
Table 1 Primer list
引物名称Primer name | 引物序列Primer sequence (5′-3′) |
---|---|
T-LjbHLH34-F | ATGGTTTCCGCGGAAAACACC |
T-LjbHLH34-R | TTAGGCAGCTGGTGGTCGGAG |
M13-F | TGTAAAACGACGGCCAGT |
pCAMBIA1307-LjbHLH34-F | |
pCAMBIA1307-LjbHLH34-R | |
pCAMBIA1307-F | AGGAAGTTCATTTCATTTGGA |
pAN580-LjbHLH34-F | |
pAN580-LjbHLH34-R | |
PAN580-F | ATGACGCACAATCCCACTATCC |
pGBKT7-LjbHLH34-F | |
pGBKT7-LjbHLH34-R | |
T7-F | TAATACGACTCACTATAGG |
qLj-actin3-F | GTATTGTTGGCCGACCTCGT |
qLj-actin3-R | AGCCTCAGTTAGAAGCACCG |
qLj-bHLH34-F | ATGCAGTTCGAGTGGTGACG |
qLj-bHLH34-R | AGACGGCAAAAATTGCCACA |
qAt-actin7-F | TCCATGAAACAACTTACAACTCCATCA |
qAt-actin7-R | CATCGTACTCACTCTTTGAAATCCACA |
qAtCAT1-F | GAGATCCCCGTGGTTTTGCT |
qAtCAT1-R | TGTGCAAACTCTCTGGGTGG |
qAtCAT3-F | AGCTTCCAGTCAATGCTCCC |
qAtCAT3-R | GTGAGACGTGGCTCCGATAG |
qAtRD22-F | TTCGTCTTCCTCTGATCTGTCTTC |
qAtRD22-R | TTTACTCCGCCTTTACCTACTTGG |
图5 转LjbHLH34基因拟南芥的鉴定M: DNA分子量标准DNA marker; -: 阴性对照Negative control; +: 阳性对照Positive control; ***表示转基因拟南芥与WT相比差异极显著(P<0.001),WT、OE-1、OE-2和OE-3分别为野生型、过表达株系1、过表达株系2和过表达株系3, 下同。*** indicates that the difference among transgenic A. thaliana and WT is extremely significant (P<0.001), WT, OE-1, OE-2 and OE-3 were wild type, overexpressed line 1, overexpressed line 2 and overexpressed line 3, the same below.
Fig.5 Identification of transgenic A. thaliana with LjbHLH34
图6 甘露醇胁迫下拟南芥的根长**表示转基因拟南芥与WT相比差异显著(P<0.01)。** indicates that the difference among transgenic A. thaliana and WT is significant (P<0.01). 下同The same below.
Fig.6 Root length of A. thaliana under D-Mannitol stress
图8 干旱胁迫下转基因拟南芥的生理指标*表示转基因拟南芥与WT相比差异显著(P<0.05)。* indicates that the difference among transgenic A. thaliana and WT is significant (P<0.05). 下同The same below.
Fig.8 Physiological indexes of transgenic A. thaliana under drought stress
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