Cloning and Activity Identification of High Efficiency Ubiquitin Promoters from Sorghum

doi:10.3969/j.issn.1000-2561.2022.12.006

Abstract

Abstract:

In the research of transgenic plants, promoter is one of the important factors affecting the efficiency of transgenic expression. Plant ubiquitin gene promoters have been widely used in monocotyledons because of its high starting efficiency, relatively low methylation degree and stable genetic traits. Among them, the Ubi-1 promoter of maize is one of the most commonly used Ubiquitin promoters. The purpose of this study is to obtain high-efficiency ubiquitin promoters of sorghum and provide a new constitutive promoter selection for the genetic transformation of sorghum and other monocotyledonous plants. In this study, multiple polyubiquitin genes were found from NCBI database, and the sequence of 3000 bp upstream of its starting codon was downloaded. According to the characteristics of Ubiquitin promoter, the sequence with 5′ UTR intron and appropriate size was selected, and the promoter was analyzed through the online promoter prediction website. Finally, two genes LOC8076096 and LOC8063786 were selected for promoter cloning, and their promoter sequences were named U1 and U5 respectively. After PCR amplification, the two promoter fragments were connected to the plant expression vector Ubi-GUS containing Gus reporter gene to obtain recombinant expression vectors U1-GUS and U5-GUS. The recombinant vector was transformed into rice and green bristlegrass by the Agrobacterium-mediated method. The positive transformed seedlings were screened by PCR. GUS staining analysis was carried out on the positive transformed seedlings to identify the promoter activities of U1 and U5. GUS staining showed that the roots, stems and leaves of all positive transformed seedlings of rice and green bristlegrass with U1 and U5 as promoters showed darker blue, which was slightly darker than that of positive transformed seedlings with maize Ubi-1 as promoter, and the blue of positive transformed seedlings with U5 as promoter was deeper than that of U1 as promoter, while non-transgenic rice and green bristlegrass seedlings could not be stained with blue at all. Therefore, the promoters U1 and U5 have the activity of constitutive strong promoters in rice and green bristlegrass, and can be used as new constitutive promoters to study the genetic transformation of sorghum, rice, green bristlegrass and other monocotyledonous plants.

Key words: sorghum, ubiquitin gene, promoter, clone, activity identification

CLC Number:

Q781

XIA Qiyu, HE Pingping, ZHANG Lili, ZHANG Yuliang, XIAO Susheng, ZHAO Hui. Cloning and Activity Identification of High Efficiency Ubiquitin Promoters from Sorghum[J]. Chinese Journal of Tropical Crops, 2022, 43(12): 2443-2452.

Figures/Tables 9

Tab. 1 Primer sequences of promoters U1 and U5

启动子Promoter	引物名称 Primer name	引物序列（5°-3°） Primer sequence (5°-3°)	预计大小 Expected size/bp
U1	U1-F	AGCTTTGTTTAAACTCAACTACCGCCATCCAACAGA	2304
U1	U1-R	CGGGGTACCCTGCAGAGAAACCAAACAAAAGGG	2304
U5	U5-F	AGCTTTGTTTAAACGTCTTGCTCCCAATATTGCTCC	2305
U5	U5-R	CGGGGTACCCTGCAAACGTCAACAAGCAAAAGC	2305

Fig. 1 Construction of recombinant expression vectors

Tab. 2 Detection primers for HPT and GUS

基因 Gene	引物名称<BREAK/>Primer name	引物序列（5<BOLD>'</BOLD>-3<BOLD>'</BOLD>）<BOLD><BREAK/></BOLD>Primer sequence (5<BOLD>'</BOLD>-3<BOLD>'</BOLD>)	大小 Size/bp
HPT	HPT -F	GAAGTGCTTGACATTGGGGAGT	472
HPT	HPT -R	AGATGTTGGCGACCTCGTATT	472
GUS	GUS -F	CCGACGCGTCCGATCACCTG	229
GUS	GUS -R	GCCCGGCTAACGTATCCA	229

Fig. 2 PCR amplification of promoters U1 and U5 M: DL2000 DNA marker.

Fig. 3 Nucleotide sequence of promoters U1 (A) and U5 (B)

Fig. 4 Bacteria liquid PCR amplification of recombinant expression vectors A: U1-GUS; B: U5-GUS; M: DL2000 DNA marker.

Fig. 5 PCR identification of NIP-U1 and NIP-U5 transformed regenerated seedlings M: DL2000 DNA marker.

Fig. 6 PCR identification of ME34-U1 and ME34-U5 transformed regenerated seedlings M: DL2000 DNA marker.

Fig. 7 GUS staining of NIP-U1, NIP-U5, ME34-U1, and ME34-U5 transformed regenerated seedlings

References 24

[1]	肖军, 石太渊, 郑秀春, 段有厚. 根癌农杆菌介导的高粱遗传转化体系的建立[J]. 杂粮作物, 2004, 24(4): 200-203.
	XIAO J, SHI T Y, ZHENG X C, DUAN Y H. Establishment of sorghum genetic transformation system with mediating Agrobacterium tumefaciens[J]. Rain Fed Crops, 2004, 24(4): 200-203. (in Chinese)
[2]	杜浩, 庄义庆, 黄萍, 杜道林. 农杆菌介导的甜高粱遗传转化研究进展[J]. 江苏农业科学, 2018, 46(4): 29-32.
	DU H, ZHUANG Y Q, HUANG P, DU D L. Research progress of agrobacterium-mediated genetic transformation in sweet sorghum[J]. Jiangsu Agricultural Sciences, 2018, 46(4): 29-32. (in Chinese)
[3]	GAO Z S, JAYARAJ J, MUTHUKRISHNAN S, CLAFLIN L, LIANG G H. Efficient genetic transformation of Sorghum using a visual screening marker[J]. Genome, 2005, 48(2): 321-333.
[4]	谢伟, 乐超银. 泛素启动子在转基因植物中的应用[J]. 三峡大学学报(自然科学版), 2007, 29(2): 176-179.
	XIE W, YUE C Y. Application of ubiquitin promoter to transgenic plants[J]. Journal of China Three Gorges University (Natural Sciences), 2007, 29(2): 176-179. (in Chinese)
[5]	TOKI S, HARA N, ONO K, ONODERA H, TAGIRI A, OKA S, TANAKA H. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice[J]. The Plant Journal, 2006, 47(6): 969-976.
[6]	赵辉, 郭静远, 孔华, 郭安平. 狗尾草胚性愈伤高效农杆菌转化体系的建立[J]. 热带作物学报, 2017, 38(6): 1106-1112.
	ZHAO H, GUO J Y, KONG H, GUO A P. The establishment of high efficient Agrobacterium transformation system of Setaria viridis[J]. Chinese Journal of Tropical Crops, 2017, 38(6): 1106-1112. (in Chinese)
[7]	CHRISTENSEN A H, SHARROCK R A, QUAIL P H. Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation[J]. Plant Molecular Biology, 1992, 18(4): 675-689. DOI PMID
[8]	郭殿京, 傅荣昭, 李文彬, 陈颖, 张晓东, 张利明, 孙勇如. 小麦中外源基因瞬间表达调控研究及兔防御素(NP-1)基因的转化[J]. 遗传学报, 1999, 26(2): 168-173.
	GUO D J, FU R Z, LI W B, CHEN Y, ZHANG X D, ZHANG L M, SUN Y R. Studies of regulation of GUS transient expression in wheat and rabbit defensin (NP-1) transformation[J]. Acta Genetica Sinica, 1999, 26(2): 168-173. (in Chinese)
[9]	CORNEJO M J, LUTH D, BLANKENSHIP K M, ANDERSON O D, BLECHL A E. Activity of a maize ubiquitin promoter in transgenic rice[J]. Plant Molecular Biology, 1993, 23(3): 567-581. DOI PMID
[10]	武亮, 卜庆云, 周明, 杨世湖, 万建民. 不同启动子驱动下马铃薯蛋白酶抑制剂PinⅡ转基因水稻的遗传、表达和对粘虫抗性分析[J]. 遗传, 2006, 28(3): 261-267.
	WU L, BU Q Y, ZHOU M, YANG S H, WAN J M. Inheritance,expression and armyworm resistance of protease inhibitorⅡ gene (PinⅡ) driven by different promoters in transgenic rice[J]. Hereditas, 2006, 28(3): 261-267. (in Chinese)
[11]	朱永生, 肖开转, 王福祥, 连玲, 何炜, 许惠滨, 魏毅东, 陈丽萍, 蒋家焕, 谢华安, 张建福. 不同启动子驱动下GL6基因表达对水稻叶表皮毛发育的影响[J]. 福建农业学报, 2019, 34(2): 139-145.
	ZHU Y S, XIAO K Z, WANG F X, LIAN L, HE W, XU H B, WEI Y D, CHEN L P, JIANG J H, XIE H A, ZHANG J F. Correlation between GL6 expression driven by varied promoters and development of epidermal hairs on leaves of rice plant[J]. Fujian Journal of Agricultural Sciences, 2019, 34(2): 139-145. (in Chinese)
[12]	郭志鸿, 王汉宁, 陶玲, 张金文, 白斌, 陈正华. ubi 启动子驱动的花生芪合酶基因表达载体的构建及玉米遗传转化初报[J]. 甘肃农业大学学报, 2004, 39(2): 117-123.
	GUO Z H, WANG H N, TAO L, ZHANG J W, BAI B, CHEN Z H. Construction of ubi promoter-based plant expression vector of stilbene synthase gene and transformation of type Ⅱ callus of maize by particle bombardment[J]. Journal of Gansu Agricultural University, 2004, 39(2): 117-123. (in Chinese)
[13]	WANG J L, JIANG J D, OARD J H. Structure, expression and promoter activity of two poly ubiquitin genes from rice[J]. Plant Science, 2000, 156(2): 201-211.
[14]	LIU D W, OARD S V, OARD J H. High transgene expression levels in sugarcane (Saccharum officinarum L.) driven by the rice ubiquitin promoter RUBQ2[J]. Plant Science, 2003, 165(4): 743-750.
[15]	CHIERA J M, BOUCHARD R A, DORSEY S L, PARK E, BUENROSTRO-NAVA M T, LING P P, FINER J J. Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system[J]. Plant Cell Reports, 2007, 26(9): 1501-1509.
[16]	GENSCHIK P, MARBACH J, UZE M, FEUERMAN M, PLESSE B, FLECK J. Structure and promoter activity of a stress and developmentally regulated polyubiquitin- encoding gene of Nicotiana tabacum[J]. Gene, 1994, 148(2): 195-202.
[17]	GARBARINO J E, BELKNAP W R. Isolation of a ubiquitin-ribosomal protein gene (ubi3) from potato and expression of its promoter in transgenic plants[J]. Plant Molecular Biology, 1994, 24(1): 119-127. DOI PMID
[18]	BINET M N, LEPETIT M, WEIL J H, TESSIER L H. Analysis of a sunflower polyubiquitin promoter by transient expression[J]. Plant Science, 1991, 79(1): 87-94.
[19]	ROLLFINKE I K, SILBER M V, PFITZNER U M. Characterization and expression of a heptaubiquitin gene from tomato[J]. Gene, 1998, 211(2): 267-276. DOI PMID
[20]	MANN D G J, KING Z R, LIU W S, JOYCE B L, PERCIFIELD R J, HAWKINS J S, LAFAYETTE P R, ARTELT B J, BURRIS J N, MAZAREI M, BENNETZEN J L, PARROTT W A, JR C N S. Switchgrass (Panicum virgatum L.) polyubiquitin gene (PvUbi1 and PvUbi2) promoters for use in plant transformation[J]. BMC Biotechnology, 2011, 11: 74.
[21]	贺飞燕, 闫建俊, 白云凤, 冯瑞云, 施俊凤. 启动子的类型及应用[J]. 山西农业科学, 2017, 45(1): 115-120.
	HE F Y, YAN J J, BAI Y F, FENG R Y, SHI J F. Types and applications of promoters[J]. Journal of Shanxi Agricultural Sciences, 2017, 45(1): 115-120.
[22]	BELIDE S, VANHERCKE T, PETRIE J R, SINGH S P. Robust genetic transformation of sorghum (Sorghum bicolor L.) using diferentiating embryogenic callus induced from immature embryos[J]. Plant Methods, 2017, 13: 109.
[23]	WANG L H, GAO L, LIU G Q, MENG R R, LIU Y L, LI J Q. An efficient sorghum transformation system using embryogenic calli derived from mature seeds[J]. Peer J, 2021, 9: e11849.
[24]	SAHA P, BLUMWALD E. Spike-dip transformation of Setaria viridis[J]. The Plant Journal, 2016, 86: 89-101.