Effects of Different Vectors and Agrobacteriumtumefaciens on Transient Expression of Alfalfa

doi:10.7525/j.issn.1673-5102.2023.06.006

Abstract

Abstract:

Alfalfa（Medicago sativa） is an important forage plants. In order to effectively express foreign genes in alfalfa， by using the green fluorescent protein（GFP） as report gene and Nicotiana benthamiana as a comparison， the effects of two different vectors（the non-replicating and the BeYDV（bean yellow dwarf virus）-based replicating vectors）， and two different Agrobacterium tumefaciens strains（LBA4404 and EHA105） on the transient expression of alfalfa leaves were investigated. The results showed that the combination of LBA4404 with the replicating vector（re-vector/LBA4404） led to effective expression of GFP in alfalfa leaves. By using LBA4404 with the replicating vector， the effects of the concentration ofre-vector/LBA4404 and incubation time after infection on the transient expression were also evaluated. It was found that with the increase of concentration of bacterial suspension or time post-infection， the expression level of GFP increased first but then decreased. In the leaves of alfalfa， the highest expression level of GFP was observed when the concentration of LBA4404 strain was 1.0（OD₆₀₀）； and the expression level of GFP reached the peak at 5-7 d after infection. Compared with Nicotiana benthamiana， achieving the highest expression level of GFP in alfalfa might need higher concentration of Agrobacterium tumefaciens and longer incubation time after infection.

Key words: Agrobacterium tumefaciens, transient expression, alfalfa

CLC Number:

S551+.7

Min SUN, Honghong YANG, Anqi WANG, Yuejing ZHANG, Xiaowei DA, Ji ZHANG, Kun SUN, Jianping WU, Hanqing FENG. Effects of Different Vectors and Agrobacteriumtumefaciens on Transient Expression of Alfalfa[J]. Bulletin of Botanical Research, 2023, 43(6): 835-845.

Figures/Tables 12

Fig.1

Fig.2

Fig.3

Fig.4

Fig.5

Fig.6

Fig.7

Fig.8

Fig.9

Fig.10

Fig.11

Fig.12

References 30

1	CHEN Q， LAI H F， HURTADO J，et al.Agroinfiltration as an effective and scalable strategy of gene delivery for production of pharmaceutical proteins［J］.Advanced Techniques in A Biology & Medicine，2013，1（1）：103.
2	CHEN Q， LAI H F.Gene delivery into plant cells for recombinant protein production［J］.BioMed Research International，2015，2015：932161.
3	PICARD K， LEE R， HELLENS R，et al.Transient gene expression in Medicago truncatula leaves via agroinfiltration［J］.Methods in Molecular Biology，2013，1069：215-226.
4	HWANG H H， YU M， LAI E M. Agrobacterium-mediated plant transformation：biology and applications［J］.The Arabidopsis Book，2017，15：e0186.
5	SHARMA A K， SHARMA M K.Plants as bioreactors：recent developments and emerging opportunities［J］.Biotechnology Advances，2009，27（6）：811-832.
6	BAI Y Q， YANG Q C， KANG J M，et al.Isolation and functional characterization of a Medicago sativa L.gene，MsLEA3-1［J］.Molecular Biology Reports，2012，39（3）：2883-2892.
7	BIAZZI E， NAZZICARI N， PECETTI L，et al.Genome-wide association mapping and genomic selection for alfalfa（Medicago sativa） forage quality traits［J］.PLoS One，2017，12（1）：e0169234.
8	SAHOO A， MANDAL A K， DWIVEDI K，et al.A cross talk between the immunization and edible vaccine：Current challenges and future prospects［J］.Life Sciences，2020，261：118343.
9	TRINH T H， RATET P， KONDOROSI E，et al.Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis［J］.Plant Cell Reports，1998，17（5）：345-355.
10	JIANG Q Z， FU C X， WANG Z Y.A unified Agrobacterium-mediated transformation protocol for alfalfa（Medicago sativa L.） and Medicago truncatula ［J］.Methods in Molecular Biology，2019，1864：153-163.
11	SAMAC D A， AUSTIN-PHILLIPS S.Alfalfa（Medicago sativa L.）［J］.Methods in Molecular Biology，2006，343：301-312.
12	KAMATÉ K， RODRIGUEZ-LLORENTE I D， SCHOLTE M，et al.Transformation of floral organs with GFP in Medicago truncatula ［J］.Plant Cell Reports，2000，19（7）：647-653.
13	CRANE C， DIXON R A， WANG Z Y. Medicago truncatula transformation using root explants［J］.Methods in Molecular Biology，2006，343：137-142.
14	ZHANG H， HUANG Q M， SU J.Development of alfalfa（Medicago sativa L.） regeneration system and Agrobacterium-mediated genetic transformation［J］.Agricultural Sciences in China，2010，9（2）：170-178.
15	WANG Z， KE Q B， KIM M D，et al.Transgenic alfalfa plants expressing the sweetpotato Orange gene exhibit enhanced abiotic stress tolerance［J］.PLoS One，2015，10（5）：e0126050.
16	ULLAH A H J， SETHUMADHAVAN K， MULLANEY E J，et al.Cloned and expressed fungal phyA gene in alfalfa produces a stable phytase［J］.Biochemical and Biophysical Research Communications，2002，290（4）：1343-1348.
17	FU C X， HERNANDEZ T， ZHOU C E，et al.Alfalfa（Medicago sativa L.）［J］.Methods in Molecular Biology，2015，1223：213-221.
18	LACROIX B， CITOVSKY V.Pathways of DNA transfer to plants from Agrobacterium tumefaciens and related bacterial species［J］.Annual Review of Phytopathology，2019，57：231-251.
19	CHEN Q， HE J Y， PHOOLCHAROEN W，et al.Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants［J］.Human Vaccines，2011，7（3）：331-338.
20	ZUBAIR M， ZAIDI S S E A， SHAKIR S，et al.Multiple begomoviruses found associated with cotton leaf curl disease in Pakistan in early 1990 are back in cultivated cotton［J］.Scientific Reports，2017，7（1）：680.
21	RAIRDAN G J， MOFFETT P.Distinct domains in the ARC region of the potato resistance protein Rx mediate LRR binding and inhibition of activation［J］.The Plant Cell，2006，18（8）：2082-2093.
22	GALÁN-ÁVILA A， GRAMAZIO P，RON M，et al.A novel and rapid method for Agrobacterium-mediated production of stably transformed Cannabis sativa L.plants［J］.Industrial Crops and Products，2021，170：113691.
23	CHINCINSKA I A.Leaf infiltration in plant science：old method，new possibilities［J］.Plant Methods，2021，17（1）：83.
24	DEGUCHI M， BOGUSH D， WEEDEN H，et al.Establishment and optimization of a hemp（Cannabis sativa L.） agroinfiltration system for gene expression and silencing studies［J］.Scientific Reports，2020，10（1）：3504.
25	BURMAN N， CHANDRAN D， KHURANA J P.A rapid and highly efficient method for transient gene expression in rice plants［J］.Frontiers in Plant Science，2020，11：584011.
26	MOËNNE-LOCCOZ Y， MAVINGUI P， COMBES C，et al.Microorganisms and biotic interactions［J］.Environmental Microbiology：Fundamentals and Applications，2014，395-444.
27	SIRIWAT W， KALAPANULAK S， SUKSANGPANOMRUNG M，et al.Unlocking conserved and diverged metabolic characteristics in cassava carbon assimilation via comparative genomics approach［J］.Scientific Reports，2018，8（1）：16593.
28	DIAMOS A G， MASON H S.Modifying the replication of geminiviral vectors reduces cell death and enhances expression of biopharmaceutical proteins in Nicotiana benthamiana leaves［J］.Frontiers in Plant Science，2019，9：1974.
29	WANG D， KHURSHID M， SUN Z M，et al.Genetic engineering of alfalfa（Medicago sativa L.）［J］.Protein And Peptide Letters，2016，23（5）：495-502.
30	吴艳菊，冷彦儒，景思荦，等.利用真空侵染法在紫花苜蓿中瞬时表达GUS基因［J］.分子植物育种，2022，20（3）：859-864.
	WU Y J， LENG Y R， JING S L，et al.Expression of GUS gene in Medicago sativa L.by vacuum infiltration［J］.Molecular Plant Breeding，2022，20（3）：859-864.

[1]	Yuejing Zhang, Ying Li, Juanjuan Wang, Hailong Pang, Lingyun Jia, Hanqing Feng. Effects of Three Kinds of Agrobacterium and Different Transformation Conditions on the Transient Expression of GFP in Nicotiana benthamiana [J]. Bulletin of Botanical Research, 2022, 42(1): 121-129.
[2]	Ren-Hua JI, Wen-Bo ZHANG, Xiao-Fei LIN, Ying-Liang BAO, Ri-Ge-Le TE, Hui-Ga BAO, Shu-Lan BAI. Subcellular Localization of the Protein Coded by the UDP-Glucose Dehydrogenase Gene from Paper Mulberry and Functional of Its Promoter 5′-end Deletion Fragment [J]. Bulletin of Botanical Research, 2020, 40(6): 932-942.
[3]	LI Mo-Ye, WANG Na, ZHOU Yu, PU Xiao-Ying, FENG Wan-Ju, LI Kai-Long. Variation Analysis of Transient Gene Expression and Growth, Physiological Indicators under Drought Stress in Expressed LbDREB Populus ussuriensis Kom. [J]. Bulletin of Botanical Research, 2016, 36(3): 409-415.
[4]	SHEN Yu-Hua;LI Wang-Feng;JIN Tai-Cheng;CHANG Qing;YIN Dong-Xu;WANG De-Li;LIU Li-Xia. Establishment and Optimization of Agrobacterium*-mediated Transformation of Suspension Embryogenic Callus of Alfalfa [J]. Bulletin of Botanical Research, 2009, 29(6): 721-727.
[5]	XU Feng-Ping;YANG Jun*;CHENG Yun-Qing;LIU Jian-Feng;AN Li-Jia. Direct DNA Transformation of Minimal Linear smGFP Gene Cassette and Its Transient Expression in Onion Epidermal Cell [J]. Bulletin of Botanical Research, 2009, 29(2): 230-233.
[6]	LI Bo, JIA Xiu-Feng, BAI Qing-Wu, TANG Yu-Hong. EFFECT OF DROUGHT STRESS ON PROLIN ACCUMULATION OF ALFALFA [J]. Bulletin of Botanical Research, 2003, 23(2): 189-191.
[7]	Wang Jin, Shi Hua-zhong, Zhou Chang, Yang Hong-yuan, Zhang Xian-long, Zhang Rong-de. TRANSIENT EXPRESSION OF GUS GENE DELIVERED INTO DE-EXINED POLLEN IN NICOTIANA TABACUM BY MICROPROJECTILE BOMBARDMENT [J]. Bulletin of Botanical Research, 1998, 18(4): 422-427.