高粱遗传转化研究进展

doi:10.3864/j.issn.0578-1752.2024.03.003

摘要/Abstract

摘要：

高粱是世界第五大粮食作物，可作为食用、饲用、酿造用和生物能源用。高粱遗传转化技术是高粱功能基因组学研究中必不可少的重要工具，同时，也可作为传统育种方法的重要补充。本文对近年来高粱遗传转化的研究进展进行总结，分析高粱遗传转化中存在的难题，并提出进一步的解决策略，以期为高粱遗传转化技术的进一步改良提供参考。通过对近年来50余篇高粱组织培养和遗传转化文献进行归纳总结。介绍用于遗传转化的高粱基因型、外植体来源、再生体系构建等方面的研究现状，对比电穿孔法、花粉管通道法、基因枪法和农杆菌介导法4种高粱遗传转化常用方法的优劣，总结遗传转化载体主要组成部分启动子、目的基因、选择标记基因和报告基因对转化效率的影响，阐述高粱遗传转化的应用现状，分析高粱遗传转化技术存在的主要瓶颈问题，研究对策措施。高粱基因型对组织培养有很大影响，以P898012和Tx430最为常用。基因枪法和农杆菌介导法是最常用的高粱遗传转化方法，且农杆菌介导法的优势逐渐显现。在载体构建中，CaMV35S启动子和ubi1启动子最为常用，抗生素抗性基因（nptII、hpt）、除草剂抗性基因（bar）和养分同化基因为常用的三大类选择标记基因。随着高粱遗传转化技术及CRISPR/Cas9介导的基因编辑技术的不断发展，一些重要农艺性状基因被成功转入高粱。但基因型依赖性强、组织培养周期长、遗传转化稳定性差是限制高粱遗传转化的主要瓶颈，通过引入形态发生调节因子，直接进行体细胞发生，缩短了组织培养周期，提高了转化效率，扩大了外植体来源，高粱遗传转化技术取得重大突破。通过引入形态发生调节因子，采用切-浸-芽（CDB）递送系统等方式可进一步改良高粱遗传转化技术，结合CRISPR/Cas9基因编辑技术的应用，必将为高粱分子育种及相关基因功能鉴定提供重要的技术支撑。

关键词: 高粱, 遗传转化, 再生体系, 转化方法, 载体

Abstract:

Sorghum is the fifth largest grain crop in the world and can be used for food, feed, brewing and bioenergy. Sorghum genetic transformation technology is an essential and important tool in the research of sorghum functional genomics and can also serve as an important complement to traditional breeding methods. In this review, we summarize the research progress of sorghum transformation in recent years, analyze the problems in sorghum genetic transformation and propose strategic solutions to them in order to provide a reference for further improvement of sorghum genetic transformation technology. By summarizing more than 50 literatures on sorghum tissue culture and genetic transformation in recent years, we introduced the current research status of sorghum genotypes, explant sources, and regeneration system construction for genetic transformation, and compared the advantages and disadvantages of four commonly used methods for sorghum genetic transformation: electroporation, pollen-mediated transformation, particle bombardment and Agrobacterium-mediated transformation, summarized the effects of the main components of genetic transformation vectors, including promoters, target genes, selective marker genes and reporter genes, on transformation efficiency, explained the current application status of sorghum genetic transformation, analyzed the main bottleneck problemns in sorghum genetic transformation technology, and studied countermeasures. Sorghum genotypes have a significant influence on tissue culture and P898012 and Tx430 are the most widely used. Gene bombardment and Agrobacterium-mediated transformation are the most commonly used methods for sorghum genetic transformation, and the advantages of Agrobacterium-mediated transformation are gradually emerging. In vector construction, CaMV35S and ubi1 are the most commonly used promoters, and antibiotic resistance genes (nptII, hpt), herbicide resistance genes (bar), and nutrient assimilation genes are the three commonly used selection markers. With the development of sorghum genetic transformation technology and CRISPR/Cas9-mediated gene editing technology, some genes with important agronomic traits have been successfully transferred into sorghum. However, strong genotype dependence, long tissue culture cycle, and poor genetic transformation stability are the main bottlenecks that limit the genetic transformation of sorghum. By introducing morphogenesis regulatory factors, somatic cell generation can be directly performed, which shortens the tissue culture cycle, improves the transformation efficiency, and expands the source of explants. This has become a major breakthrough in sorghum genetic transformation technology. The use of morphogenesis regulatory factors and adoption of cut-dip-budding (CDB) delivery system can further improve the sorghum genetic transformation technology. Combined with the application of CRISPR/Cas9 gene editing technology, they will surely provide an important technical basis for the sorghum molecular breeding and gene function identification.

Key words: sorghum, transformation technology, regenerative system, transformation method, vector

韩立杰, 才宏伟. 高粱遗传转化研究进展[J]. 中国农业科学, 2024, 57(3): 454-468.

HAN LiJie, CAI HongWei. Progress on Genetic Transformation of Sorghum[J]. Scientia Agricultura Sinica, 2024, 57(3): 454-468.

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图/表 1

表1

高粱遗传转化研究进展"

转化方法 Transformation method	基因型 Genotype	外植体 Explant	载体 Vector	选择标记 Selectable marker	启动子 Promoter	筛选剂及筛选压 Screening agent and screening pressure	报告基因 Report gene	转基因检测 Transgenic detection	转化效率 Transformation efficiency (%)	参考文献 References
电穿孔法 Electroporation	-	原生质体 Protoplast	p35SCAT/ pCopa-CAT	-	CaMV35S copia promoter of Drosophila	氯霉素 Chloramphenicol	-	Scintillation counting	-	[30]
电穿孔法 Electroporation	NK300	原生质体 Protoplast	pBI221.1	nptⅡ	CaMV35S	100 mg·L^-1卡那霉素 Kanamycin	GUS	Gel blot hybridization	-	[31]
电穿孔法 Electroporation	Sorghum vulgare (cv Grazer)	细胞悬浮液 Cell suspension	pBC1/pNGI	nptⅡ/hpt	adh1/CaMV35S	500 mg·L^-1卡那霉素Kanamycin/50 mg·L^-1 潮霉素Hygromycin	GUS	GUS assay/ Gel blot hybridization	-	[32]
花粉管通道法 Pollen-mediated transformation	TX622B	花柱 Style	-	nptⅡ	-	100 mg·L^-1卡那霉素 Kanamycin	-	丝黑穗病鉴定/聚丙烯酰氨凝胶电泳 Head smut disease detection/ polyacrylamide gel electrophoresis	6.80	[33]
花粉管通道法 Pollen-mediated transformation	A（2）V4A/A（2）V4B	花粉 Pollen	pBI121	nptⅡ	CaMV35S	卡那霉素 Kanamycin	GUS	PCR/Southern blot	-	[34]
基因枪法 Particle bombardment	P898012	幼胚 Immature embryo	pPHP620/ pPHP687	bar	CaMV35S	3 mg·L^-1 bialophos	GUS	Southern blot/GUS assay	0.29	[7]
基因枪法 Particle bombardment	SRN39	幼穗 Immature infloresences	pPHP620	bar	CaMV35S	3 mg·L^-1 bialophos	GUS	Southern blot/GUS assay	2.61	[35]
基因枪法 Particle bombardment	Tx430	幼胚 Immature embryo	-	bar	CaMV35S	1-2 mg·L^-1 basta	-	Southern blot/Western blot/ PCR	0.09	[36]
基因枪法 Particle bombardment	M35-1/SA281/QL41/P898012	幼胚 Immature embryo	pAHC20	bar	ubi1/act1/ CaMV35S	2 mg·L^-1 bialophos	GFP	Southern blot/荧光显微镜 Southern blot/Fluorescence microscope	1.00	[9]
基因枪法 Particle bombardment	RTx430	幼胚 Immature embryo	pAHC20/ pAct1-D	bar	ubi1/act1	5 mg·L^-1 PPT	GUS	Southern blot/GUS assay	0.18	[37]
基因枪法 Particle bombardment	Tx430/C401/ CO25	幼胚 Immature embryo	pPZP200/ pUC18	hpt	ubi1/CaMV35S/HBT/act1/adh1	-	GUS/GFP	荧光显微镜/GUS assay Fluorescence microscope/ GUS assay	-	[5]
基因枪法 Particle bombardment	恢复系 Restorer line 5-27/115	幼穗 Immature infloresences	pKUB	hpt	-	20 mg·L^-1潮霉素 Hygromycin	GUS	PCR/Southern blot/ GUS assay	-	[38]
基因枪法 Particle bombardment	214856	幼胚/芽尖 Immature embryo/Shoot tip	pAct1-D/ pAHC25	nptⅡ	ubi1/adh1/act1D/ CaMV35S	25-100 mg·L^-1遗传霉素Geneticin	GUS	PCR/Southern blot/ GUS assay	1.30	[39]
基因枪法 Particle bombardment	BTx623	芽尖 Shoot tip	pJS108/ pmpiC1cry1Ac	bar	mpiC1	1-2.05 mg·L^-1 basta	GUS	PCR/Southern blot/ GUS assay	1.50	[28]
基因枪法 Particle bombardment	32个品种 Varieties	幼胚 Immature embryo	pPH1687	hpt	ubi1	40 mg·L^-1潮霉素 Hygromycin	LUC	Southern blot/荧光素酶测定 Southern blot/Luciferase camera assay	0.09	[40]
基因枪法 Particle bombardment	P898012	幼胚 Immature embryo	pAHC25/ pNOV3604	bar/pmi	ubi1	9 g·L^-1甘露糖 Mannose+12 g·L^-1 麦芽糖Maltose	GUS	PCR/Southern blot	0.77	[41]
基因枪法 Particle bombardment	Tx430	幼胚 Immature embryo	pUKN/pGEM-Ubi-gfp	nptⅡ	ubi1	30 mg·L^-1遗传霉素 Geneticin（G418）	GFP	PCR/Southern blot/荧光显微镜 PCR/Southern blot/ Fluorescence microscope	20.70	[6]
基因枪法 Particle bombardment	Hiro1	成熟胚 Mature embryo	pCAMBIA1301/p35S::SbGW2-GFP	hpt	ubi/CaMV35S	10 mg·L^-1 潮霉素 Hygromycin	GFP	PCR/荧光显微镜 PCR/Fluorescence microscope	12.31	[42]
基因枪法 Particle bombardment	Tx430	幼胚 Immature embryo	pBSV003	nptⅡ	ubi1	25-35 mg·L^-1遗传霉素 Geneticin（G418）	GUS	PCR/GUS assay	27.20-46.60	[23]
基因枪法 Particle bombardment	IS4698	成熟胚 Mature embryo	pMD18T-UBI-NPTII/pMD18T-UBI-GUS	nptⅡ	ubi1	30 mg·L^-1遗传霉素 Geneticin（G418）	GUS	PCR/GUS assay	13.33	[43]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404	P898012/ PHI391	幼胚 Immature embryo	pSB1/pSB11	bar	ubi1	5 mg·L^-1 PPT	GUS	Southern blot	2.10	[8]
农杆菌介导法 Agrobacterium-mediated transformation EHA105/EHA101/AGL1	Tx430/C401/ Wheatland	幼胚/叶子 Immature embryo/Leaf	pPZP200/ pUC18	-	ubi1/CaMV35S/ HBT/act1/adh1	-	GUS/GFP	荧光显微镜/GUS assay Fluorescence microscope/ GUS assay	-	[5]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404	P898012	幼胚 Immature embryo	pTOK233	hpt	CaMV35S	15 mg·L^-1潮霉素 Hygromycin	GUS	Southern blot/GUS assay	0.80-3.50	[20]
农杆菌介导法 Agrobacterium-mediated transformation EHA101	C401/ Pioneer8505	幼胚 Immature embryo	pPZP201	pmi	ubi1	1%-2%甘露糖 Mannose	GFP	Southern blot/Western blot/荧光显微镜 Southern blot/Western blot/ Fluorescence microscope	2.88-3.30	[44]
农杆菌介导法 Agrobacterium-mediated transformation EHA101	Tx430/C401/ Pioneer8505	幼胚 Immature embryo	pPZP201	-	ubi1	-	GFP	Southern blot/Western blot/荧光显微镜 Southern blot/Western blot/ Fluorescence microscope	1.00-3.00	[45]
农杆菌介导法 Agrobacterium-mediated transformation NTL4	Tx430/C2-97	幼胚 Immature embryo	pPTN290	nptⅡ	ubi1	10 mg·L^-1遗传霉素 Geneticin/20 mg·L^-1 巴龙霉素Paromomycin	GUS	Southern blot/GUS assay	0.30-4.50	[21]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404	Sensako 85/ 1191	幼胚 Immature embryo	pCAMBIA1301	hpt	CaMV35S	5 mg·L^-1潮霉素 Hygromycin	GUS	Southern blot/GUS assay	5.00	[25]
农杆菌介导法 Agrobacterium-mediated transformation EHA101/LBA4404	P898012/Tx430/296B/C401	幼胚 Immature embryo	pPZP201	pmi	ubi1	1%-2%甘露糖 Mannose	GFP	PCR/Western blot/荧光显微镜 PCR/Western blot/ Fluorescence microscope	8.30	[46]
农杆菌介导法 Agrobacterium-mediated transformation EHA105	恢复系 Restorer line 115/5-27/9198/R011 保持系 Maintainer line ICS21B/21B	幼穗 Immature infloresences	pKUB	hpt	CaMV35S/ ubi1	50 mg·L^-1潮霉素 Hygromycin	GUS	RT-PCR/Southern blot/ Western blot	1.90	[10]
农杆菌介导法 Agrobacterium-mediated transformation EHA101	P898012	幼胚 Immature embryo	pZY102/pZY101 -TC2/pZY101-SKRS	bar	CaMV35S/ CZ19B1	2.5 mg·L^-1草铵膦 Glufosinate-ammonium	-	PCR/Southern blot	0.40-0.70	[47]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404/EHA105	CO25/ TNS586	幼胚 Immature embryo	pMKURF2/ pCAMBIAG11/ pCAMBIARC7	bar/hpt	ubi1	1-2 mg·L^-1 bialophos/ 10-25 mg·L^-1潮霉素 Hygromycin	GUS	Western blot	1.60-2.70	[48]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404	M 35-1	成熟胚/幼苗/叶基/幼穗 Mature embryo/Young seedling/Leaf base/ Immature infloresences	pKU352NA	hpt	ubi1	20 mg·L^-1潮霉素 Hygromycin	SGFPS65T	Inverse PCR	4.28	[17]
农杆菌介导法 Agrobacterium-mediated transformation NTL4	P898012/ RTX430	幼胚 Immature embryo	pCAMBIA1305.2/pCAM-Ubi-gus	hpt	ubi1	20 mg·L^-1潮霉素 Hygromycin	GUS	PCR/Southern blot	2.40	[24]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404	APK1	芽尖 Shoot tip	pCAMBIA1305.1	hpt	CaMV35S	5 mg·L^-1潮霉素 Hygromycin	GUS	PCR/Southern blot	1.20-3.90	[49]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404/AGL1	TX430	幼胚 Immature embryo	pSB1/pSB11	moPAT/ pmi	ubi1	5-10 mg·L^-1 PPT/ 12.5 g·L^-1 甘露糖Mannose	DSRED	qPCR	10.00-33.00	[22]
农杆菌介导法 Agrobacterium-mediated transformation AGL1/EHA101/GV3101	P898012	幼胚 Immature embryo	pZY102/ pFGC5941/ pFGC161	bar	MAS/ubi1/ CaMV35S	2.5 mg·L^-1草铵膦 Glufosinate-ammonium	GUS	PCR/Southern blot	14.00	[50]
农杆菌介导法 Agrobacterium-mediated transformation EHA105	U68/Tx623B	成熟胚 Mature embryo	pCAMBIA1305.1 /pCUbi1390	hpt	ubi1	-	GFP	PCR/Western blot	4.00	[29]
农杆菌介导法 Agrobacterium-mediated transformation EHA105	P898012	幼胚 Immature embryo	PHP78891	-	ubi1	-	GFP	PCR/Southern blot	6.20	[14]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404 Thy-	Tx430/Macia/ Malisor84-7/ Tegemeo	幼胚 Immature embryo	pPHP38332/ pPHP78152/ pPHP78233/ pPHP81561/ pPHP70444	pmi/nptⅡ/ PTXD	ubi1	12.5 g·L^-1甘露糖 Mannose/250 mg·L^-1 遗传霉素Geneticin (G418)/300 mg·L^-1 Phi	YFP	qPCR	6.00-29.00	[13]
农杆菌介导法 Agrobacterium-mediated transformation GV2260	Tx430	幼胚 Immature embryo	pEKH2	hpt	CaMV35S	15 mg·L^-1潮霉素 Hygromycin	GUS	PCR/Southern blot	1.90	[51]
农杆菌介导法 Agrobacterium-mediated transformation AGL1/EHA101	P898012/ Btx623	幼胚 Immature embryo	PHP78891	-	ubi1/Rab17/ Nos	-	GFP	荧光显微镜 Fluorescence microscope	-	[15]
农杆菌介导法 Agrobacterium-mediated transformation EHA105	BTx623	幼胚 Immature embryo	pMDC32- 35S-GFP	hpt	CaMV35S	2-5 mg·L^-1潮霉素 Hygromycin	GFP	PCR/RT-PCR/荧光显微镜 PCR/RT-PCR/Fluorescence microscope	0.73	[12]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404	Tx430/ Tx2737	幼穗/芽尖 Immature infloresences/ Shoot tip	pBI121	nptⅡ	CaMV35S	50-100 mg·L^-1 卡那霉素Kanamycin	GUS	PCR/GUS assay	4.20-14.30	[11]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404 Thy-	Tx430/Tx623/ Tx2752/Macia/ Malisor 84-7/ Tegemeo	幼胚 Immature embryo	pPHP79066/ pPHP81814	Hra/nptⅡ	Pltp/Axig1	250 mg·L^-1遗传霉素 Geneticin (G418)	ZS- YELLOW1	荧光显微镜 Fluorescence microscope	17.10-69.70	[16]
农杆菌介导法 Agrobacterium-mediated transformation LBA4404 TD Thy-	Tx430	叶片 Leaf	PHP96037	Hra	ubi1/Nos	-	-	-	35.00-37.00	[18]

表1

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