植物研究 ›› 2022, Vol. 42 ›› Issue (5): 855-865.doi: 10.7525/j.issn.1673-5102.2022.05.017
收稿日期:
2021-09-09
出版日期:
2022-09-20
发布日期:
2022-09-15
通讯作者:
张力鹏
E-mail:nknanhai@163.com
作者简介:
王宏鹏(1994—),男,硕士研究生,主要从事植物分子细胞遗传学研究。
Hongpeng WANG, Yidan LI, Yao WANG, Xiaoyu TAN, Chengbin CHEN, Lipeng ZHANG()
Received:
2021-09-09
Online:
2022-09-20
Published:
2022-09-15
Contact:
Lipeng ZHANG
E-mail:nknanhai@163.com
About author:
WANG Hongpeng(1994—),male,postgraduate,major in plant molecular cytogenetics.
摘要:
以菊叶薯蓣为研究材料,采用RT-PCR技术克隆获得DcPMK基因,进行生物信息学、组织特异性和诱导表达分析,并构建DcPMK的诱饵载体以筛选拟南芥酵母文库中互作蛋白,为深入研究菊叶薯蓣萜类物质的合成积累提供一定理论基础。结果表明:DcPMK基因开放阅读框大小为1 536 bp(GenBank登录号MZ171241),编码511个氨基酸。蛋白质序列比对分析发现DcPMK与近源种基因序列一致性达88.85%,具有1个保守的ATP结合位点Gly-X-Gly-XX-Ala。N-J进化树显示DcPMK与海枣(Phoenix dactylifera)等单子叶植物的遗传距离较近。HPLC结果显示菊叶薯蓣皂素主要在根茎中积累,qRT-PCR结果表明DcPMK基因在各组织中均有表达,在老茎中表达量最高,根茎中表达量最低;水杨酸诱导后皂素含量的变化与DcPMK表达量的变化趋势吻合:随着叶片中皂素含量提高,DcPMK上调表达。与阴性对照相比,DcPMK基因没有自激活性和细胞毒性,并筛选到27个互作明显的拟南芥基因,如生长发育相关基因AtKCR1(AT1G67730)、AtRPS9M(AT3G49080)、AtASY4(AT2G33793),非生物与生物逆境相关基因AtVDAC2(AT5G67500)、AtVDAC3(AT5G15090)、AtRH8(AT4G00660)及花色素苷积累相关基因AtPHR2(AT2G47590)等。以上结果说明DcPMK能够参与菊叶薯蓣的萜类物质合成,并通过蛋白质-蛋白质相互作用的方式广泛参与其生长发育和胁迫响应等代谢途径。
中图分类号:
王宏鹏, 李一丹, 汪耀, 谭晓宇, 陈成彬, 张力鹏. 菊叶薯蓣DcPMK基因克隆及互作蛋白筛选[J]. 植物研究, 2022, 42(5): 855-865.
Hongpeng WANG, Yidan LI, Yao WANG, Xiaoyu TAN, Chengbin CHEN, Lipeng ZHANG. Gene Cloning and Interaction Proteins Screening of DcPMK in Dioscorea composite[J]. Bulletin of Botanical Research, 2022, 42(5): 855-865.
表1
引物名称及序列
引物 Primer | 引物序列 Primer sequence(5′—3′) | 用途 Strategy |
---|---|---|
DcPMK-F | ATGGCGGTTGTGGCTTCAGC | 基因克隆 gene cloning |
DcPMK-R | CTAGTTGATATTGATGGAAGAAATAGACG | |
DcPMK-BDCE-F | tatggccatggaggccgaattcATGGCGGTTGTGGCTTCAGC | 酵母双杂交引物 Yeast two-hybrid primers |
DcPMK-BDCE-R | gccgctgcaggtcgacggatccCCTAGTTGATATTGATGGAAGAA | |
DcTUB7-qF | AGACAACATCAACCCTGGACT | 内参基因引物 Internal reference primers |
DcTUB7-qR | GAGGCTGAGAGCAACATGAAT | |
DcPMK-qF | 荧光定量PCR引物 Primers for qRT-PCR | |
DcPMK-qR |
表2
DcPMK 基因互作基因信息
阳性菌编号 No. | 拟南芥基因号 Gene ID | 基因大小Length /bp | 基因名称 Name | 基因注释信息 Description |
---|---|---|---|---|
4 | ATCG00740 | 990 | RPOA | RNA聚合酶α亚基 RNA polymerase alpha subunit. |
5,31 | AT5G06340 | 1 035 | ATNUDX27 | nudix水解酶27 Nudix hydrolase homolog 27 |
6 | AT3G51130 | 1 233 | unknown | 跨膜蛋白 Transmembrane protein. |
7 | AT1G67730 | 957 | ATKCR1 | 编码β-酮酰基还原酶(KCR),该酶催化VLCFA延伸过程中的第一次还原反应 Beta-ketoacyl reductase(KCR),which catalyzes the first reduction during VLCFA elongation |
8,44 | AT2G47590 | 1 344 | PHR2 | 光解酶/蓝光光感受器PHR2 mRNA photolyase/blue light photoreceptor PHR2 mRNA |
9,40 | AT3G10910 | 546 | DAFL1 | RING/U-box超家族蛋白 RING/U-box superfamily protein |
11,19,29,41 | AT3G60770 | 456 | unknown | 核糖体蛋白S13/S15 Ribosomal protein S13/S15 |
14 | AT4G16555 | 525 | unknown | 类HSP20伴侣 HSP20-like chaperone |
16 | AT2G05325 | 536 | unknown | 新的转录区;在根,心皮等中检测到 Novel transcribed region;detected in root,carpel,and so on |
17 | AT2G43235 | 1 314 | unknown | 磷酸核糖甲酰甘氨酸脒合酶 phosphoribosylformylglycinamidine synthase |
22 | AT3G60570 | 795 | EXPB5 | β-扩张蛋白成员 member of BETA-EXPANSINS |
23 | AT3G55330 | 693 | PPL1 | 促进光系统Ⅱ超级复合物的组装,并在波动光下优化植物的垂体 Facilitates the assembly of the photosystem Ⅱ supercomplexes and optimizes plant pitness under fluctuating light |
24 | AT3G49080 | 1 293 | RPS9M | 功能丧失导致配子致死 Loss of function results in gametophyte lethality |
26 | AT3G47080 | 1 548 | unknown | 类超家族蛋白-TPR TPR-like superfamily protein |
27,28 | AT4G00660 | 1 518 | RH8 | 类RNA解旋酶-8 RNA helicase-like 8 |
30,34,39 | AT2G43130 | 645 | ARA4 | 其涉及细胞内囊泡运输 Which are implicated in intracellular vesicular traffic |
32 | AT1G78170 | 666 | unknown | 泛素连接酶E3 E3 ubiquitin-protein ligase |
33 | AT5G20290 | 669 | unknown | 核糖体蛋白S8e家族蛋白 Ribosomal protein S8e family protein |
35 | AT2G18770 | 783 | unknown | 含有P环的核苷三磷酸水解酶超家族蛋白 P-loop containing nucleoside triphosphate hydrolases superfamily protein |
36 | AT1G55120 | 1 785 | FRUCT5 | 植物的生长发育中起作用 Plays a role in the growth and development of plants |
38 | AT5G15090 | 825 | VDAC3 | 编码依赖电压的阴离子通道。它们参与细胞器和胞质溶胶之间的代谢物交换 Encodes a voltage-dependent anion channel. They are involved in metabolite exchange between the organelle and the cytosol |
42 | AT5G16710 | 1 088 | DHAR3 | 调节细胞质中抗坏血酸-谷胱甘肽循环的氧化还原状态 Modulate redox states of ascorbate-glutathione cycle in the cytosol |
43 | AT5G67500 | 912 | VDAC2 | 它们参与细胞器和胞质溶胶之间的代谢物交换 They are involved in metabolite exchange between the organelle and the cytosol |
45 | AT5G47190 | 1 009 | PRPL19 | 核糖体蛋白L19家族蛋白 Ribosomal protein L19 family protein |
46 | AT5G41910 | 561 | MED10A | 介体复合物亚基10 Mediator complex,subunit Med10 |
47 | AT2G33793 | 639 | ASY4 | 编码突触前染色体轴的蛋白质成分。突变体在减数分裂重组中显示出定量缺陷 Encodes a protein component of the pre-synaptic chromosome axis;Mutants show quantitative defects in meiotic recombination |
48 | AT2G33800 | 912 | SCA1 | 等位基因sca1-rps5在质体16S rRNA加工中表现出缺陷,并导致光合积累的减少 The sca1-rps5 allele exhibits defects in plastid 16S rRNA processing and a resulting decrease in accumulation of photosynthetic |
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