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

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

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

• Genetic & Breeding · Germplasm Resources · Molecular Biology • Previous Articles Next Articles

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 Online:2023-10-25 Published:2023-10-30

Abstract

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

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.

Figures/Tables 10

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

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

Fig. 2 Schematic diagram of the amiRNA precursor

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

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

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.

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.

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.

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）.

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

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[2]	PAN Jiajia, ZHANG Dongmei, MENG Jian, GAO Sunan, ZHU Kaijie, LIU Junwei, LI Guohuai. Optimization and Validation of PpPDS Gene Silencing Induced by Prunus Necrotic Ring Spot Virus in Prunus persica [J]. Acta Horticulturae Sinica, 2023, 50(7): 1587-1600.
[3]	GONG Danmin, CAI Linzhi, DING Ren, MA Yanyan, WANG Wanxin, XIONG Xingyao, HU Xinxi. Effects of Different Potassium Fertilizers on Growth and Absorption and Accumulation of Mineral Elements and Cadmium in Potato [J]. Acta Horticulturae Sinica, 2023, 50(6): 1332-1342.
[4]	TAN Jie, LIU Jiangang, LIU Ju, JIAN Yinqiao, LI Guangcun, JIN Liping, XU Jianfei. Cultivated Substrates Evaluation for in-situ Observation of Potato Tubers Using Computed Tomography [J]. Acta Horticulturae Sinica, 2023, 50(5): 1085-1094.
[5]	YIN Jian, ZHU Xijian, WU Yaoyao, LI Canhui, ZHANG Jinzhe. Targets Prediction and Function Analysis of Potato miR319 Gene Family [J]. Acta Horticulturae Sinica, 2023, 50(3): 583-595.
[6]	ZHU Jie, LI Lu, SUN Mingfei, GAO Meina, LIANG Bowen, XU Jizhong, LI Zhongyong. Effects of Different Growth Type Scions on Root Architecture of Self-rooted Young Apple Trees [J]. Acta Horticulturae Sinica, 2023, 50(10): 2183-2191.
[7]	WANG Kuan, QI Lipan, WU Guili, FENG Yan, WANG Lei, YIN Jiang, LUO Yating, WANG Yan, LIU Chang, GONG Xuechen, and WANG Haijun. A New Potato Cultivar‘Beifang 002’of Early Maturity and Good Yield [J]. Acta Horticulturae Sinica, 2022, 49(S2): 141-142.
[8]	QI Lipan, FENG Yan, WANG Lei, YIN Jiang, WANG Kuan, LUO Yating, GONG Xuechen, LIU Chang, and WANG Yan. A New Potato Cultivar‘Beifang 004’ [J]. Acta Horticulturae Sinica, 2022, 49(S2): 143-144.
[9]	LI Yanshan, SUI Qijun, JIANG Wei, YANG Qiongfen, and BAI Jianming. A New Potato Cultivar‘Yunshu 113’for Both Fresh Consumption and Starch Extraction [J]. Acta Horticulturae Sinica, 2022, 49(S2): 145-146.
[10]	ZOU Xue, DING Fan, LIU Lifang, YU Hankaizong, CHEN Nianwei, and RAO Liping. A New Purple Potato Cultivar‘Mianziyu 1’ [J]. Acta Horticulturae Sinica, 2022, 49(S1): 93-94.
[11]	YAN Wenyuan, QIN Junhong, DUAN Shaoguang, XU Jianfei, JIAN Yinqiao, JIN Liping, LI Guangcun. The Effect of Water-nitrogen Coupling on Potato Photosynthesis,Tuber Formation and Quality [J]. Acta Horticulturae Sinica, 2022, 49(7): 1491-1504.
[12]	QIU Keli, WANG Yumin, HE Jinling, YU Hong, PAN Haifa, SHENG Yu, XIE Qingmei, CHEN Hongli, ZHOU Hui, ZHANG Jinyun. Identification of Peach Laccase Family Genes and Function Analysis of PpLAC21 [J]. Acta Horticulturae Sinica, 2022, 49(6): 1351-1362.
[13]	QI Lipan, LI Yue, WANG Lei, FENG Yan, WANG Kuan, YIN Jiang, GUO Huachun. Anatomical Observation on the Graft Union Between Potato and Wolfberry [J]. Acta Horticulturae Sinica, 2022, 49(4): 868-874.
[14]	DAI Wenshan, WU Yue, WANG Min. Preliminary Study on Mechanisms of Resistance to Citrus Canker of Kumquat FcRGA1 [J]. Acta Horticulturae Sinica, 2022, 49(11): 2325-233.
[15]	CHEN Jingyi, FANG Boping, ZHANG Xiongjian, HUANG Lifei, WANG Zhangying, and LUO Zhongxia. A New Leaf-vegetable Sweet Potato Cultivar‘Guangcaishu 5’ [J]. Acta Horticulturae Sinica, 2021, 48(S2): 2861-2862.