马铃薯miR397的克隆及靶基因筛选

doi:10.3969/j.issn.1004-1524.2022.06.05

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (6): 1141-1151.DOI: 10.3969/j.issn.1004-1524.2022.06.05

马铃薯miR397的克隆及靶基因筛选

李文翔¹(), 王芳¹^,²^,³^,⁴^,⁵^,⁶^,^*(), 王舰¹^,²^,³^,⁴^,⁵^,⁶

1.青海大学农牧学院,青海西宁 810016
2.青海省农林科学院,青海西宁 810016
3.青藏高原生物技术教育部重点实验室,青海西宁 810016
4.青海省马铃薯育种重点实验室,青海西宁 810099
5.青海大学西北马铃薯教育部工程研究中心,青海西宁 810016
6.青海大学省部共建三江源生态与高原农牧业国家重点实验室,青海西宁 810016

收稿日期:2022-02-22 出版日期:2022-06-25 发布日期:2022-06-30
通讯作者: 王芳
作者简介:*王芳,E-mail: qhwf324@163.com
李文翔（1996—）,男,甘肃通渭人,硕士研究生,从事马铃薯遗传育种研究。E-mail: 1724132162@qq.com
基金资助:
青海省科技厅项目(2020-NK-170);国家现代农业产业技术体系(CARS-10);宁波市2025重大种业专项(2019B10017)

Cloning and target gene screening of miR397 in potato

LI Wenxiang¹(), WANG Fang¹^,²^,³^,⁴^,⁵^,⁶^,^*(), WANG Jian¹^,²^,³^,⁴^,⁵^,⁶

1. College of Agriculture and Animal HUsbandry, Qinghai University, Xining 810016, China
2. Qinghai Academy of Agriculture and Forestry Sciences, Xining 810016, China
3. Key Laboratory of Qinghai-Tibetan Plateau Biotechnology of Ministry of Education, Xining 810016, China
4. Key Laboratory of Potato Breeding of Qinghai, Xining 810099, China
5. Engineering Research Center of Potato in Northwest Region, Ministry of Education, Xining 810016, China
6. State key Laboratory of Plateau Ecology and Agriculture, Xining 810016, China

Received:2022-02-22 Online:2022-06-25 Published:2022-06-30
Contact: WANG Fang

摘要/Abstract

摘要：

miR397广泛参与植物逆境胁迫响应。本研究从马铃薯叶片中分离出miR397的前体序列,克隆得到StmiR397基因,对潜在靶基因进行预测,采用实时荧光定量PCR（qRT-PCR）方法对低温不同表现型材料进行表达模式分析。结果表明：马铃薯miR397前体序列为81 bp,能形成稳定的茎环结构,成熟序列位于5′端,上游调控区含有温度响应等多种逆境应答作用元件。低温处理下,StmiR397在耐低温材料DR-2叶中上调表达,根中表达无差异,而在低温敏感材料费乌瑞它叶中表达无差异,根中上调表达。通过对预测的10个靶基因进行表达模式分析,初步筛选出反向抑制靶基因氧化还原酶结构域蛋白、锌指家族蛋白和正向激活靶基因磷酸果糖激酶家族蛋白、生长素诱导的β-葡萄糖苷酶,为进一步研究StmiR397与靶基因的调控网络提供了一定参考。

关键词: 马铃薯, 低温胁迫, 靶基因

Abstract:

miR397 is widely involved in plant stress response. In this study, the precursor sequence of miR397 was isolated from potato leaves, the StmiR397 was cloned and its potential target genes were predicted. The qRT-PCR was used to analyze the expression patterns of different low-temperature phenotypic materials, the results showed: the length of precursor sequence of potato miR397 was 81 bp, which could form stable stem-loop structure, and the mature sequence was located at the 5' end. The upstream regulatory region contained various stress response elements such as temperature response. Under low temperature treatment, the expression of StmiR397 was up-regulated in the leaves of the low-temperature resistant material DR-2, and the expression showed no difference in the roots, while the expression of StmiR397 showed no difference in the leaves of the low-temperature sensitive material Favorite, and the expression was up-regulated in the roots. According to the expression patterns analyses of the 10 predicted target genes, oxidoreductase-like domain-containing protein gene, zinc knuckle (CCHC-type) family protein gene of the reverse inhibitory target genes and the positive activation target genes 6-phosphate 1-phosphotransferase subunit alpha gene and auxin-induced beta-glucosidase gene were initially screened. This study provides a certain reference for further research on the regulatory network of StmiR397 and target genes.

Key words: potato, low temperature stress, target gene

中图分类号:

S532

李文翔, 王芳, 王舰. 马铃薯miR397的克隆及靶基因筛选[J]. 浙江农业学报, 2022, 34(6): 1141-1151.

LI Wenxiang, WANG Fang, WANG Jian. Cloning and target gene screening of miR397 in potato[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1141-1151.

图/表 9

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引物名称 Primer name	引物序列 Primer sequence（5’→3’）
StmiR397-F	ATTGAGTGCAGCGTTGATGAC
StmiR397-R	TGATTGAGTGTAGCGTTGATGAAG
LAC7-F	CATTTGGATGTTCACTTGCCTTGGG
LAC7-R	CTGGTGGAGGAGGAGGTAGCATAG
M1C3Q2-F	ACAAGTCATTACTACTGCCGCTTCC
M1C3Q2-R	TAGTCCTGCTGCTCCTCCGTTATAC
ICE-F	GATGGACAGGGCTTCAATCTTAGGG
ICE-R	CGGCGTGGACTCCAGTTCATTATG
M1BIR1-F	TCGGCTACATTCACCAACAACACC
M1BIR1-R	ACCGCTGAAAGGATTGCTACACTG
M0ZH35-F	CGGTGGACAGTGATGGGACATTC
M0ZH35-R	AGCCTAAACGAGAACCAGCAAGATC
M1BHR0-F	GATCCAGATAACCGCCGCAAGAG
M1BHR0-R	TGCCCTTCCTGTCCACAATGTTTAC
LAC3-F LAC3-R	CACCACTACCACTGCCATACTTGAG TGGCTGTATCATTGAAGGCTGGAAG
M1CV13-F		TACCGAGCATCCAGCGAAGAGG
M1CV13-R		GAACCACCCAGAAAACCAAGCAAAG
M1C9X3-F		GACCATCCAGCGACTCTTCACTTG
M1C9X3-R		TGCCTTTCCAGTGCTTGTAGTTCTC
M1AZQ5-F		AGGGAAATGGGAGAGGACAAGAGAG
M1AZQ5-R		CTGTATTCGCTGGTCTGCTGATCTG
M0ZW04-F		AACCAATCTTCCTCATCGGCAACC
M0ZW04-R		GCAGCGAAATTACGGCTTGATGG
miR397-F		CGCGATTGAGTGCAGCGTT
StA F		AGATGCTTACGCTGGATGGAATGC
StA R		TTCCGGTGTGGTTGGATTCTGTTC

引物名称 Primer name	引物序列 Primer sequence（5’→3’）
StmiR397-F	ATTGAGTGCAGCGTTGATGAC
StmiR397-R	TGATTGAGTGTAGCGTTGATGAAG
LAC7-F	CATTTGGATGTTCACTTGCCTTGGG
LAC7-R	CTGGTGGAGGAGGAGGTAGCATAG
M1C3Q2-F	ACAAGTCATTACTACTGCCGCTTCC
M1C3Q2-R	TAGTCCTGCTGCTCCTCCGTTATAC
ICE-F	GATGGACAGGGCTTCAATCTTAGGG
ICE-R	CGGCGTGGACTCCAGTTCATTATG
M1BIR1-F	TCGGCTACATTCACCAACAACACC
M1BIR1-R	ACCGCTGAAAGGATTGCTACACTG
M0ZH35-F	CGGTGGACAGTGATGGGACATTC
M0ZH35-R	AGCCTAAACGAGAACCAGCAAGATC
M1BHR0-F	GATCCAGATAACCGCCGCAAGAG
M1BHR0-R	TGCCCTTCCTGTCCACAATGTTTAC
LAC3-F LAC3-R	CACCACTACCACTGCCATACTTGAG TGGCTGTATCATTGAAGGCTGGAAG
M1CV13-F		TACCGAGCATCCAGCGAAGAGG
M1CV13-R		GAACCACCCAGAAAACCAAGCAAAG
M1C9X3-F		GACCATCCAGCGACTCTTCACTTG
M1C9X3-R		TGCCTTTCCAGTGCTTGTAGTTCTC
M1AZQ5-F		AGGGAAATGGGAGAGGACAAGAGAG
M1AZQ5-R		CTGTATTCGCTGGTCTGCTGATCTG
M0ZW04-F		AACCAATCTTCCTCATCGGCAACC
M0ZW04-R		GCAGCGAAATTACGGCTTGATGG
miR397-F		CGCGATTGAGTGCAGCGTT
StA F		AGATGCTTACGCTGGATGGAATGC
StA R		TTCCGGTGTGGTTGGATTCTGTTC

元件Element	序列 Sequence	功能Function
TGACG-motif	TGACG	参与茉莉酸响应的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness
TCA-element	CCATCTTTTT	参与水杨酸响应的顺式作用元件 cis-acting element involved in salicylic acid responsiveness
TATC-box	TATCCCA	参与赤霉素响应的顺式作用元件 cis-acting element involved in gibberellin-responsiveness
ABRE	ACGTG	参与脱落酸响应的顺式作用元件 cis-acting element involved in the abscisic acid responsiveness
TATA-box	ATATAT	核心启动子元件Core promoter element
TATA-box	TATA	核心启动子元件Core promoter element
TATA-box	TATAAA	核心启动子元件Core promoter element
TATA-box	TATAA	核心启动子元件Core promoter element
CGTCA-motif	CGTCA	参与茉莉酸响应的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness
P-box	CCTTTTG	赤霉素响应元件Gibberellin-responsive element
CAAT-box	CAAAT	启动子和增强子区域的常见顺式作用元件 Common cis-acting element in promoter and enhancer regions
MRE	AACCTAA	参与光响应MYB结合位点MYB binding site involved in light responsiveness
3-AF1 binding site	TAAGAGAGGAA	光响应元件Light responsive element
TCT-motif	TCTTAC	光响应元件的一部分Part of a light responsive element
I-box	gGATAAGGTG	光响应元件的一部分Part of a light responsive element
GT1-motif	GGTTAA	光响应元件Light responsive element
ARE	AAACCA	厌氧诱导所必需的顺式作用调节元件 cis-acting regulatory element essential for the anaerobic induction
G-Box	CACGTT	参与光响应的顺式作用调节元件 cis-acting regulatory element involved in light responsiveness
TC-rich repeats	GTTTTCTTAC	参与防御和应激反应的顺式作用元件 cis-acting element involved in defense and stress responsiveness
CCAAT-box	CAACGG	MYBHv1结合位点MYBHv1 binding site
HSE	CTCC	参与温度响应的顺式作用元件 cis-acting element involved in temperature responsiveness

元件Element	序列 Sequence	功能Function
TGACG-motif	TGACG	参与茉莉酸响应的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness
TCA-element	CCATCTTTTT	参与水杨酸响应的顺式作用元件 cis-acting element involved in salicylic acid responsiveness
TATC-box	TATCCCA	参与赤霉素响应的顺式作用元件 cis-acting element involved in gibberellin-responsiveness
ABRE	ACGTG	参与脱落酸响应的顺式作用元件 cis-acting element involved in the abscisic acid responsiveness
TATA-box	ATATAT	核心启动子元件Core promoter element
TATA-box	TATA	核心启动子元件Core promoter element
TATA-box	TATAAA	核心启动子元件Core promoter element
TATA-box	TATAA	核心启动子元件Core promoter element
CGTCA-motif	CGTCA	参与茉莉酸响应的顺式作用调节元件 cis-acting regulatory element involved in the MeJA-responsiveness
P-box	CCTTTTG	赤霉素响应元件Gibberellin-responsive element
CAAT-box	CAAAT	启动子和增强子区域的常见顺式作用元件 Common cis-acting element in promoter and enhancer regions
MRE	AACCTAA	参与光响应MYB结合位点MYB binding site involved in light responsiveness
3-AF1 binding site	TAAGAGAGGAA	光响应元件Light responsive element
TCT-motif	TCTTAC	光响应元件的一部分Part of a light responsive element
I-box	gGATAAGGTG	光响应元件的一部分Part of a light responsive element
GT1-motif	GGTTAA	光响应元件Light responsive element
ARE	AAACCA	厌氧诱导所必需的顺式作用调节元件 cis-acting regulatory element essential for the anaerobic induction
G-Box	CACGTT	参与光响应的顺式作用调节元件 cis-acting regulatory element involved in light responsiveness
TC-rich repeats	GTTTTCTTAC	参与防御和应激反应的顺式作用元件 cis-acting element involved in defense and stress responsiveness
CCAAT-box	CAACGG	MYBHv1结合位点MYBHv1 binding site
HSE	CTCC	参与温度响应的顺式作用元件 cis-acting element involved in temperature responsiveness

名称Name	基因描述Gene description	靶向mRNA ID Target mRNA	起始位置Target start	终止位置Target end	得分Score	靶向比对序列Target aligned Fragment	碱基配对Match	miRNA比对序列miRNA aligned fragment	功能Function
LAC7	漆酶7 Laccase-7-like	Soltu.DM.07G016640.1	676	696	1	AUCAUCAACGCUGCACUCAAU	::::::::::::::::::::	CAGUAGUUGCGACGUGAGUUA	催化活性、结合Catalytic activity, binding
LAC3	漆酶3 Laccase-3-like	Soltu.DM.06G032550.1	433	453	1	GUGAUCAACGCUGCACUCAAU	:: ::::::::::::::::::	CAGUAGUUGCGACGUGAGUUA	催化活性、结合Catalytic activity, binding
M1BIR1（PPR）	含五肽重复序列的蛋白质 Pentatricopeptide repeat-containing protein	Soltu.DM.09G030600.1	1021	1040	6	GUCAUAACUGCUGCA-UCAAA	::::: :.:::::: ::::	CAGUAGUUGCGACGUGAGUUA	生物过程Biological process
M0ZH35 （APs1）	天冬氨酸蛋白酶1 Aspartic proteinase 1	Soltu.DM.01G035170.1	674	694	6	GUAAGAAAUGCUACACUCAAU	:: : ::.::: ::::::::	CAGUAGUUGCGACGUGAGUUA	天冬氨酸型内肽酶活性、水解酶活性、非生物刺激响应Aspartic endopeptidase activity, hydrolase activity, abiotic stimulation response
M1BHR0（CCHC）	锌指家族蛋白 Zinc knuckle (CCHC-type) family protein	Soltu.DM.05G006320.2	18	37	4	GUCAUCAAUGUUGC-CUCAAU	::::::::.:.::: ::::::	CAGUAGUUGCGACGUGAGUUA	mRNA结合、翻译调节活性、锌离子结合、核酸结合mRNA binding, translation regulatory activity, zinc ion binding, nucleic acid binding
M0ZW04 (Olp)	氧化还原酶结构域蛋白 Oxidoreductase-like domain-containing protein	Soltu.DM.02G024460.1	304	323	4.5	GUAAUUU-CGC UGCACUCAAA	:: ::. ::::::::::::	CAGUAGUUGCGACGUGAGUUA	氧化还原酶活性Oxidoreductase activity
M1AZQ5 (CTC7)	含五肽重复序列的蛋白质 Pentatricopeptide repeat-containing protein	Soltu.DM.08G011800.1	455	476	5	GUCAUCAACGUCUGCAGUUAAU	:::::::::: ::::: :.:::	CAGUAGUUGCGACGUGAGUUA	蛋白质结合、mRNA结合Protein binding, mRNA binding
M1C9X3 （MIS12）	动力蛋白复合物MIS12 homologue	Soltu.DM.03G028560.1	177	197	5	UUCAUCAACGAGGCACUCAAU	::::::::: :::::::::	CAGUAGUUGCGACGUGAGUUA	细胞过程、细胞成分组织、细胞分裂Cell process, cell composition, tissue, cell division
M1CV13 （PFP）	磷酸果糖激酶家族蛋白6-phosphate 1-phosphotransferase subunit alpha	Soltu.DM.12G004610.1	1839	1859	6	GUCCUGAAAGCUGCACUUAGU	::: : :: ::::::::.:.:	CAGUAGUUGCGACGUGAGUUA	6-磷酸果糖激酶活性、 ATP结合、激酶活性、光合作用Fructokinase-6-phosphate activity, ATP binding, kinase activity, photosynthesis
M1C3Q2（GHs1）	生长素诱导的β-葡萄糖苷酶 Auxin-induced beta-glucosidase	Soltu.DM.11G017090.1	330	349	6	GUGAACAAUGCUGCAC-CAGU	:: : :::.::::::: ::.:	CAGUAGUUGCGACGUGAGUUA	水解酶活性、分解代谢过程、碳水化合物代谢过程Hydrolase activity, catabolic process, carbohydrate metabolism process

马铃薯miR397的克隆及靶基因筛选

Cloning and target gene screening of miR397 in potato

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