草业学报 ›› 2023, Vol. 32 ›› Issue (7): 49-60.DOI: 10.11686/cyxb2022425
王少鹏1(), 刘佳1(), 洪军2, 林积圳2, 张义2, 史昆1, 王赞1()
收稿日期:
2022-10-27
修回日期:
2022-11-22
出版日期:
2023-07-20
发布日期:
2023-05-26
通讯作者:
王赞
作者简介:
E-mail: zanwang@cau.edu.cn基金资助:
Shao-peng WANG1(), Jia LIU1(), Jun HONG2, Ji-zhen LIN2, Yi ZHANG2, Kun SHI1, Zan WANG1()
Received:
2022-10-27
Revised:
2022-11-22
Online:
2023-07-20
Published:
2023-05-26
Contact:
Zan WANG
摘要:
干旱是影响植物生长发育及产量的重要环境因素,PPR(pentatricopeptide repeats)家族蛋白在植物生长发育以及胁迫响应等生理过程中都具有重要作用。本研究在中苜1号紫花苜蓿中克隆到MsPPR1,利用病毒介导的基因沉默技术和烟草异源表达,在中苜1号紫花苜蓿和烟草中验证了MsPPR1在抗旱性中的功能。结果显示,MsPPR1开放阅读框包含3213 bp,编码1070个氨基酸,相对分子量为121.65 kDa,具有多个PPR重复结构域,定位于细胞质,是典型的PPR蛋白家族成员。MsPPR1主要表达在叶中,其次是茎和根,花中最少;其表达量受自然干旱、甘露醇和脱落酸处理的诱导。通过病毒诱导基因沉默技术在紫花苜蓿中降低了MsPPR1的表达,干旱胁迫下,沉默植株更加萎蔫,相对含水量显著降低,相对电解质渗透率显著升高,显著降低了紫花苜蓿的抗旱性。在烟草中异源超表达MsPPR1,显著增强了转基因烟草的抗旱性,干旱胁迫下,丙二醛含量显著降低,脯氨酸含量增加。以上结果均表明MsPPR1是紫花苜蓿抗旱性的正调控因子,本研究为紫花苜蓿抗旱分子育种提供了候选基因。
王少鹏, 刘佳, 洪军, 林积圳, 张义, 史昆, 王赞. 紫花苜蓿MsPPR1基因的克隆及抗旱功能分析[J]. 草业学报, 2023, 32(7): 49-60.
Shao-peng WANG, Jia LIU, Jun HONG, Ji-zhen LIN, Yi ZHANG, Kun SHI, Zan WANG. Cloning and function analysis of MsPPR1 in alfalfa under drought stress[J]. Acta Prataculturae Sinica, 2023, 32(7): 49-60.
引物名称Primers | 引物序列Primer sequence (5'-3') |
---|---|
MsPPR1-F | ATGTTGTTCTCCTCAACAAAACCC |
MsPPR1-R | TCAGTTCATAGACTCTGTCATGTCTGA |
MsPPR1-GFP-F | TTAATTAAATGTTGTTCTCCTCAACAAAA |
MsPPR1-GFP-R | GGCGCGCCGTTCATAGACTCTGTC |
pBI121-MsPPR1-F | GAGAACACGGGGGACTCTAGAATGTTGTTCTCCTCAAC |
pBI121-MsPPR1-R | AAGGGACTGACCACCCGGGGAGTTCATAGACTCTGT |
pTRV2-MsPPR1-F | TGAGTAAGGTTACCGAATTCTGGGTTTGTGGGTATGTTGA |
pTRV2-MsPPR1-R | GTGAGCTCGGTACCGGATCCTGAGAACTCATCCGCCTCCT |
MsPPR1-qPCR-F | GTTTCTAGTTCCGTGGTTTCGGCGT |
MsPPR1-qPCR-R | AAACTTCATCAACCCTCCCCAACTT |
MsActin-qPCR-F | CAAAAGATGGCAGATGCTGAGGAT |
MsActin-qPCR-R | CATGACACCAGTATGACGAGGTCG |
表 1 试验所用引物
Table 1 Primers used in the study
引物名称Primers | 引物序列Primer sequence (5'-3') |
---|---|
MsPPR1-F | ATGTTGTTCTCCTCAACAAAACCC |
MsPPR1-R | TCAGTTCATAGACTCTGTCATGTCTGA |
MsPPR1-GFP-F | TTAATTAAATGTTGTTCTCCTCAACAAAA |
MsPPR1-GFP-R | GGCGCGCCGTTCATAGACTCTGTC |
pBI121-MsPPR1-F | GAGAACACGGGGGACTCTAGAATGTTGTTCTCCTCAAC |
pBI121-MsPPR1-R | AAGGGACTGACCACCCGGGGAGTTCATAGACTCTGT |
pTRV2-MsPPR1-F | TGAGTAAGGTTACCGAATTCTGGGTTTGTGGGTATGTTGA |
pTRV2-MsPPR1-R | GTGAGCTCGGTACCGGATCCTGAGAACTCATCCGCCTCCT |
MsPPR1-qPCR-F | GTTTCTAGTTCCGTGGTTTCGGCGT |
MsPPR1-qPCR-R | AAACTTCATCAACCCTCCCCAACTT |
MsActin-qPCR-F | CAAAAGATGGCAGATGCTGAGGAT |
MsActin-qPCR-R | CATGACACCAGTATGACGAGGTCG |
图1 MsPPR1的生物信息学分析A: MsPPR1的克隆Clone of MsPPR1, M: DNA分子量标准DNA marker; B: MsPPR1蛋白质二级结构的预测Prediction of the secondary structure of MsPPR1; C: MsPPR1的保守结构域The conserved structural domains of MsPPR1.
Fig.1 Bioinformatic analysis of the deduced MsPPR1
图2 多物种中PPR家族蛋白的系统进化树分析图为MsPPR1与其他植物PPR蛋白进化树分析。The figure shows the phylogenetic tree analysis of MsPPR1 and other plant PPR proteins. XP_020220752.1: 木豆C. cajan; XP_007138785.1: 菜豆P. vulgaris; XP_027903061.1: 豇豆V. unguiculata; XP_003595043.1: 蒺藜苜蓿M. truncatula; XP_003547448.1: 大豆G. max; XP_019415098.1: 狭叶羽扇豆L. angustifolius; XP_004487972.1: 鹰嘴豆C. arietinum; XP_004239478.1: 番茄S. lycopersicum; XP_045788966.1: 红三叶T. pratense; KAF8102602.1: 白芥S. alba; NP_001078759.1: 拟南芥A. thaliana; XP_006401224.1: 盐芥E. salsugineum; MCD7471248.1: 曼陀罗D. stramonium; XP_019228083.1: 烟草N. benthamiana; KAF2949520.1: 粳稻O. sativa Japonica Group; PWZ31433.1: 玉米Z. mays; XP_002457610.1: 高粱S. bicolor.
Fig.2 Phylogenetic tree analysis of PPR family proteins in multiple species
图4 紫花苜蓿MsPPR1相对表达量分析A: 不同组织中的表达The relative expression of MsPPR1 in the indicated tissues of alfalfa; B: 自然干旱处理下的表达The relative expression of MsPPR1 under nature drought; C: 甘露醇处理下的表达The relative expression of MsPPR1 under mannitol (200 mmol·L-1); D: 脱落酸处理下的表达The relative expression of MsPPR1 under ABA (100 μmol·L-1).不同小写字母表示处理之间差异显著(P<0.05)。Different lowercase letters indicate significant differences among treatments (P<0.05). 下同The same below.
Fig.4 Analysis of relative expression of MsPPR1 in alfalfa
图5 病毒诱导沉默下MsPPR1表达量及紫花苜蓿干旱表型A: MsPPR1在沉默植株及对照植株中的表达量The relative expression of MsPPR1 in silent plants and control plants; B: 对照和干旱情况下0和20 d植株情况Plants in 0 and 20 days under control and drought conditions; C: 干旱处理情况下0、8、20 d植株叶片相对含水量Leaf relative leaf water content of plants in 0, 8 and 20 days under drought treatment。pTRV-water、pTRV-drought、pTRV-MsPPR1-water和pTRV-MsPPR1-drought分别为对照植株正常浇水、对照植株干旱处理、沉默MsPPR1植株正常浇水和沉默MsPPR1植株干旱处理。pTRV-water, pTRV-drought, pTRV-MsPPR1-water, and pTRV-MsPPR1-drought represent control plants water treatment, control plants drought treatment, silent MsPPR1 plants water treatment, silent MsPPR1 plants drought treatment respectively. D: 干旱处理情况下0、8、20 d植株相对电解质渗透率Relative electrolyte leakage of plants in 0, 8 and 20 days after drought treatment.
Fig.5 Expression level of MsPPR1 and phenotype under drought stress in alfalfa by virus-induced gene silencing
图6 不同株系烟草MsPPR1相对表达量及其表型A: 转基因烟草的验证The verification of genetically modified tobacco, M:DNA分子量标准DNA marker; B: 转基因烟草中MsPPR1相对表达量The relative expression of MsPPR1 in transgenic tobacco. WT、OE-1、OE-2、OE-3、OE-4、OE-5和OE-6 分别为野生型、过表达株系1、过表达株系2 、过表达株系3、过表达株系4、过表达株系5和过表达株系6,下同。WT,OE-1,OE-2, OE-3 OE-4,OE-5 and OE-6 were wild type,overexpressed line 1,overexpressed line 2, overexpressed line 3, overexpressed line 4, overexpressed line 5 and overexpressed line 6, the same below; C: 干旱处理情况下转基因烟草的表型The phenotype of transgenic tobacco under drought treatment.
Fig.6 Relative expression and phenotype of MsPPR1 in different tobacco strains
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