利用人工miRNA沉默StIAA22对马铃薯根系构型的影响

doi:10.16420/j.issn.0513-353x.2022-0761

园艺学报 ›› 2023, Vol. 50 ›› Issue (10): 2091-2103.doi: 10.16420/j.issn.0513-353x.2022-0761

• 遗传育种·种质资源·分子生物学 • 上一篇下一篇

利用人工miRNA沉默StIAA22对马铃薯根系构型的影响

杨江伟¹^,², 段小琴², 张飞燕², 张丽³, 邓玉荣², 王晓凤², 郑智勇², 张宁², 司怀军¹^,²^,^*()

¹ 甘肃农业大学省部共建干旱生境作物学国家重点实验室，兰州 730070
² 甘肃农业大学生命科学技术学院，兰州 730070
³ 宁夏农林科学院农业生物技术研究中心，银川 750002

收稿日期:2023-05-12 修回日期:2023-07-13 出版日期:2023-10-25 发布日期:2023-10-30
通讯作者: *（E-mail：hjsi@gsau.edu.cn）
基金资助:
国家自然科学基金项目(31860400); 甘肃农业大学青年研究生指导教师扶持基金项目(GAU-QDFC-2021-15); 省部共建干旱生境作物学国家重点实验室基金项目(GSCS-2017-6); 甘肃省科技重大专项计划项目(GZGG-2021-6)

Root Architecture Alteration by Down-regulation of the StIAA22 Gene Using Artificial MicroRNA in Potato

YANG Jiangwei¹^,², DUAN Xiaoqin², ZHANG Feiyan², ZHANG Li³, DENG Yurong², WANG Xiaofeng², ZHENG Zhiyong², ZHANG Ning², SI Huaijun¹^,²^,^*()

¹ State Key Laboratory of Aridland Crop Science，Gansu Agricultural University，Lanzhou 730070，China
² College of Life Science and Technology，Gansu Agricultural University，Lanzhou 730070，China
³ Agricultural Biotechnology Research Center，Ningxia Academy of Agricultural and Forestry Sciences，Yinchuan 750002，China

Received:2023-05-12 Revised:2023-07-13 Published:2023-10-25 Online:2023-10-30
Contact: *（E-mail：hjsi@gsau.edu.cn）

摘要/Abstract

摘要：

通过检测26个StIAA家族基因在马铃薯根系发育过程中的表达发现：18个StIAA基因在根系成熟后的表达量比发育初期显著上调，8个StIAA基因变化不显著。利用人工microRNA（artificial microRNA，amiRNA）技术对上调表达最为显著的StIAA22做进一步的功能研究：构建干扰表达载体pBI121-amiRIAA，通过根癌农杆菌介导法转入马铃薯栽培品种‘Dèsirèe’。经定量PCR检测，StIAA22在所有转基因株系中的表达均受到明显抑制；其根系形态构型与非转基因植株差异明显，根系生长受到抑制，根长明显变短，侧根数量增加，根系生物量减少。以上结果表明StIAA22在调节马铃薯根系形态建成中起关键作用。

关键词: 马铃薯, StIAA22, 人工miRNA, 基因沉默, 根系形态

Abstract:

Quantitative real-time PCR（qRT-PCR）were used to analyze the expression levels of 26 StIAA genes during potato root development，the results showed that 18 StIAAs were obviously up-regulated between the early period and the mature period of potato development，expression level of other eight StIAAs are not obvious. In addition，artificial microRNA（amiRNA）were used to futher functional study of StIAA22 that is one of the most significant up-regulated StIAAs. Interference vector of pBI121-amiRIAA was constructed，and transferred into the potato genome mediated by Agrobactium tumefacienses to obtain the transgenic plants. The qRT-PCR analysis showed that the expression of StIAA22 was obviously restrained in all transgenic plant lines. Also，the transgenic lines showed a drastic change in root architecture compared with that in control plants by suppressing StIAA22 expression，such as severe reduction in the length of root，an increase in number of lateral roots，and a decrease in biomass of potato roots. The results showed that StIAA22 had a key role in modulating potato root architecture.

Key words: potato, StIAA22, artificial miRNA, gene silencing, root architecture

杨江伟, 段小琴, 张飞燕, 张丽, 邓玉荣, 王晓凤, 郑智勇, 张宁, 司怀军. 利用人工miRNA沉默StIAA22对马铃薯根系构型的影响[J]. 园艺学报, 2023, 50(10): 2091-2103.

YANG Jiangwei, DUAN Xiaoqin, ZHANG Feiyan, ZHANG Li, DENG Yurong, WANG Xiaofeng, ZHENG Zhiyong, ZHANG Ning, SI Huaijun. Root Architecture Alteration by Down-regulation of the StIAA22 Gene Using Artificial MicroRNA in Potato[J]. Acta Horticulturae Sinica, 2023, 50(10): 2091-2103.

图/表 10

图1 马铃薯StIAA22基因结构及amiR-IAA设计

Fig. 1 The structures and amiR-IAA sequence of potato StIAA22 gene

表1 巢式PCR构建amiR-IAA载体所需引物

Table 1 Primers for construction of amiR-IAA vector using overlapping PCR

引物名称 Primers name	引物（5′-3′） Primers sequence
amiR-IAA-I	GATAGACCCTTTATAGGCTTCGCTCTCTCTTTTGTATTCC
amiR-IAA-II	GAGCGAAGCCTATAAAGGGTCTATCAAAGAGAATCAATGA
amiR-IAA-III	GAGCAAAGCCTATAATGGGTCTTTCACAGGTCGTGATATG
amiR-IAA-IV	GAAAGACCCATTATAGGCTTTGCTCTACATATATATTCCT

图2 人工miRNA（amiRNA）前体产生的示意图

Fig. 2 Schematic diagram of the amiRNA precursor

表2 产生amiRNA前体的标准反应体系

Table 2 Standard reaction system of amiRNA precursor

PCR反应 PCR reaction	正向引物 Forward oligo	反向引物 Reverse oligo	模板 Template	产物长度/bp Length of PCR product
(a)	A	Ⅳ	pRS300	272
(b)	Ⅲ	Ⅱ	pRS300	171
(c)	Ⅰ	B	pRS300	298
(d)	A	B	(a) + (b) + (c)	701

图3 马铃薯StIAA基因在根系不同发育阶段的表达模式 * 表示P < 0.05时差异显著，** 表示P < 0.01 时差异显著。

Fig. 3 Expression pattern of StIAAs in potato root development * indicates signifificant difference（P < 0.05），** indicates signifificant difference（P < 0.01）.

图4 马铃薯StIAA22基因amiRNA载体的构建 A：d片段测序结果，红色线条代表pRS300载体上ath-miR319a的成熟序列和其反向序列经重叠PCR扩增后替换为amiRIAA成熟序列和其反向序列；B：pBI121-amiRIAA表达载体的结构。

Fig. 4 Construction of suppress expression vector of StIAA22 with amiRNA A：Sequencing of the d fragment，the red lines represent mature sequence of miR319a and its reverse sequence on original pRS300 vector，and amiRIAA mature sequence and its reverse sequence after PCR amplification；B：Construction of pBI121-amiRIAA expression vector.

图5 转基因植株NPTII基因的PCR检测 M：DL2 000 marker；V：载体质粒；CK：未转基因植株；T1 ~ T10：转基因植株。

Fig. 5 PCR detection of NPTII gene in the transgenic plants M：DL2 000 marker；V：Vector；CK：Non-transgenic plants；T1-T10：Transgenic plants.

图6 RLM-5° RACE验证amiRIAA对StIAA22基因mRNA的降解位点括号内的分数表示所有测序克隆中显示断裂位点的克隆比例。

Fig. 6 RLM-5° RACE verification of StIAA22 gene mRNA cleavage sites generated by amiRIAA Fraction within parentheses means the proportions of 5°RACE clones showing these cleavage sites out of all sequenced clones.

图7 人工miRNA抑制了马铃薯StIAA22的表达 CK：未转基因植株；T1 ~ T10：转基因植株，* 表示P < 0.05 时差异显著，** 表示P < 0.01 时差异显著。

Fig. 7 Artifificial miRNAs silence the expression of the StIAA22 in potato CK：Non-transgenic plants；T1-T10：Transgenic plants，* indicates signifificant difference（P < 0.05），** indicates signifificant difference（P < 0.01）.

图8 转基因马铃薯植株（T1，T2和T9）与非转基因植株（CK）的表型特征

Fig. 8 Phenotypic characteristics of transgenic potato plants（T1，T2 and T9）and non-transgenic（CK） * P < 0.05，** P < 0.01.

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利用人工miRNA沉默StIAA22对马铃薯根系构型的影响

Root Architecture Alteration by Down-regulation of the StIAA22 Gene Using Artificial MicroRNA in Potato

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利用人工miRNA沉默StIAA22对马铃薯根系构型的影响

Root Architecture Alteration by Down-regulation of the StIAA22 Gene Using Artificial MicroRNA in Potato

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