酸枣愈伤组织转化体系构建及在ZjBRC1调控ZjYUCCA表达中的应用

doi:10.16420/j.issn.0513-353x.2021-0292

摘要/Abstract

摘要：

以来源于同一株酸枣实生苗的茎段、叶片、子叶和胚轴等组织为外植体试材,筛选到适合子叶、胚轴和叶片诱导愈伤组织的培养基为MS + 1.0 mg · L^-12,4-D + 0.4 mg · L^-1 TDZ;利用农杆菌介导法,当浸染液浓度OD₆₀₀为0.6 ~ 0.8、浸染时间为30 min时,酸枣叶片、子叶、胚轴和茎段愈伤组织遗传转化率分别为31.8%、25.6%、24.5%和23.8%。利用叶片诱导的愈伤组织转化体系,获得了调控分枝发育关键因子ZjBRC1融合表达嵌合抑制子SRDX和35S::GFP空载体对照的转基因愈伤组织。荧光定量qRT-PCR分析显示,与35S::GFP转基因愈伤组织相比,ZjBRC1-SRDX下调生长素合成基因ZjYUCCA7/10-3/10-4的表达,而上调ZjYUCCA2/4/6表达。综上,本研究中建立和优化了枣愈伤组织诱导和转化体系。

关键词: 枣, 愈伤组织, 遗传转化, ZjBRC1, 基因表达

Abstract:

The stem segment,leaves,cotyledon and hypocotyls from the same sour jujube（Ziziphus jujuba Mill. var. spinosa）were used as the explants to induce callus. The optimum callus induction was obtained from cotyledon,hypocotyls and leaves in MS media supplemented with 1.0 mg · L^-1 2,4- dichlorophenoxyacetic acid（2,4-D）and 0.4 mg · L^-1 thidiazuron（TDZ）. Furthermore,the callus transformation efficiency was 31.8% in leaves,25.6% in cotyledons,24.5% in hypocotyls and 23.8% in stem segments,respectively,after inoculating callus in the Agrobacterium infection solution with OD₆₀₀= 0.6-0.8 for 30 min. In addition,ZjBRC1,which was a homologue of CYC/TB1 TCP transcription factor BRC1 and the key repressor to control shoot branching,were fused to the N terminus of SRDX repressor domain. 35S::GFP and 35S::ZjBRC1-SRDX transgenic calli were obtained via the leaf-induced callus transformation system. qRT-PCR analysis showed that auxin biosynthetic genes ZjYUCCA7/10-3/10-4 were down-regulated while ZjYUCCA2/4 /6 were up-regulated in the 35S::ZjBRC1-SRDX transgenic calli.

Key words: jujube, callus, genetic transformation, ZjBRC1, gene expression

中图分类号:

S665.1

李亚梅, 马福利, 张山奇, 黄锦秋, 陈梦婷, 周军永, 孙其宝, 孙俊. 酸枣愈伤组织转化体系构建及在ZjBRC1调控ZjYUCCA表达中的应用[J]. 园艺学报, 2022, 49(4): 749-757.

LI Yamei, MA Fuli, ZHANG Shanqi, HUANG Jinqiu, CHEN Mengting, ZHOU Junyong, SUN Qibao, SUN Jun. Optimization of Jujube Callus Transformation System and Application of ZjBRC1 in Regulating ZjYUCCA Expression[J]. Acta Horticulturae Sinica, 2022, 49(4): 749-757.

图/表 8

参考文献 21

[1]	Braun N, de Saint Germain A, Pillot J P, Boutet-Mercey S, Dalmais M, Antoniadi I, Li X, Maia-Grondard A, Le Signor C, Bouteiller N, Luo D, Bendahmane A, Turnbull C, Rameau C. 2012. The pea TCP transcription factor PsBRC 1 acts downstream of strigolactones to control shoot branching. Plant Physiology, 158 (1):225-238. doi: 10.1104/pp.111.182725 pmid: 22045922
[2]	Cui Yan-hong, Wang Huan, Dou Su-han, Zhang Ya-dong, Pang Xiao-ming, Li Ying-yue. 2018. Establishment of SPDS system for leaf genetic transformation in Ziziphus jujuba Mill. var. spinosa mediated by Agrobacterium. Molecular Plant Breeding, 16 (20):6710-6717. (in Chinese)
	崔艳红, 王欢, 豆苏含, 张亚东, 庞晓明, 李颖岳. 2018. 农杆菌介导酸枣叶片遗传转化SPDS基因体系的建立. 分子植物育种, 16 (20):6710-6717.
[3]	Domagalska M A, Leyser O. 2011. Signal integration in the control of shoot branching. Nature Reviews Molecular Cell Biology, 12 (4):211-221. doi: 10.1038/nrm3088 URL
[4]	Dong Q L, Liu D D, An X H, Hu D G, Yao Y X, Hao Y J. 2011. MdVHP1 encodes an apple vacuolar H⁺-PPase and enhances stress tolerance in transgenic apple callus and tomato. Journal of Plant Physiology, 168 (17):2124-2133. doi: 10.1016/j.jplph.2011.07.001 URL
[5]	Dun E A, de Saint Germain A, Rameau C, Beveridge C A. 2012. Antagonistic action of strigolactone and cytokinin in bud outgrowth control. Plant Physiology, 158 (1):487-498. doi: 10.1104/pp.111.186783 URL
[6]	Feng Xiao-dong, Chen Guo-liang, Qiao Jing, Chang Hai-fei. 2014. Study on Agrobacterium tumefacien-mediated genetic transformation system for callus of Ziziphus jujuba cv. Junzao. Journal of Yanan University(Natural Science Edition), 33 (1):54-57. (in Chinese)
	冯晓东, 陈国梁, 乔璟, 常海飞. 2014. 根癌农杆菌介导的骏枣愈伤组织遗传转化体系的研究. 延安大学学报(自然科学版), 33 (1):54-57.
[7]	Gu X, Meng H, Qi G, Zhang J. 2008. Agrobacterium-mediated transformation of the winter jujube(Ziziphus jujuba Mill.). Plant Cell,Tissue and Organ Culture, 94:23-32. doi: 10.1007/s11240-008-9383-z URL
[8]	Guo M X, Zhang Z R, Cheng Y W, Li S N, Shao P Y, Yu Q, Wang J J, Xu G, Zhang X T, Liu J J, Hou L L, Liu H X, Zhao X S. 2020a. Comparative population genomics dissects the genetic basis of seven domestication traits in jujube. Horticulture Research, 7 (1):89. doi: 10.1038/s41438-020-0312-6 URL
[9]	Guo M X, Zhang Z R, Li S P, Lian Q, Fu P C, He Y L, Qiao J X, Xu K K, Liu L P, Wu M Y, Du Z R, Li S N, Wang J J, Shao P Y, Yu Q, Xu G, Li D K, Wang Y K, Tian S, Zhao J, Feng X, Li R Q, Jiang W K, Zhao X S. 2020b. Genomic analyses of diverse wild and cultivated accessions provide insights into the evolutionary history of jujube. Plant Biotechnology Journal, 19 (3):517-531. doi: 10.1111/pbi.13480 URL
[10]	Liu M J, Zhao J, Cai Q L, Liu G C, Wang J R, Zhao Z H, Liu P, Dai L, Yan G J, Wang W J, Li X S, Chen Y, Sun Y D, Liu Z G, Lin M J, Xiao J, Chen Y Y, Li X F, Wu B, Ma Y, Jian J B, Yang W, Yuan Z, Sun X C, Wei Y L, Yu L L, Zhang C, Liao S G, He R J, Guang X M, Wang Z, Zhang Y Y, Luo L H. 2014. The complex jujube genome provides insights into fruit tree biology. Nature Communications, 5 (1):5315. doi: 10.1038/ncomms6315 URL
[11]	Luo Zai-qi, Guo Hui-li, Yang Ya-dong, Yang Ming-feng, Ma Lan-qing, Wang You-nian. 2015. Agrobacterium-mediated transformation of resveratrol synthase gene(PcPKS5)into Huping Jujube(Ziziphus jujuba). Scientia Silvae Sinicae, 51 (10):101-109. (in Chinese)
	罗在柒, 郭辉力, 杨亚东, 杨明峰, 马兰青, 王有年. 2015. 农杆菌介导虎杖芪合酶基因遗传转化壶瓶枣的研究. 林业科学, 51 (10):101-109.
[12]	Ma F L, Huang J Q, Yang J, Zhou J Y, Sun Q B, Sun J. 2020. Identification,expression and miRNA targeting of auxin response factor genes related to phyllody in the Witches’ broom disease of jujube. Gene, 746:144656. doi: 10.1016/j.gene.2020.144656 URL
[13]	Ma Fuli, Mu Wenlei, Zhou Junyong, Lu Lijuan, Sun Qibao, Wang Haiyan, Sun Jun. 2020. Activity and function analysis of MdRDACO1flanking the retrotransposon Mdsolo-LTR1 insertion site in‘Red Delicious’apple spur mutants. Acta Horticulturae Sinica, 47 (8):1429-1437. (in Chinese)
	马福利, 穆文磊, 周军永, 陆丽娟, 孙其宝, 王海燕, 孙俊. 2020. ‘元帅’苹果短枝变异逆转座子插入位点临近基因MdRDACO1的表达及功能分析. 园艺学报, 47 (8):1429-1437.
[14]	Ma Q Y, Li S X, Bi C W, Hao Z D, Sun C R, Ye N. 2017. Complete chloroplast genome sequence of a major economic species,Ziziphus jujuba (Rhamnaceae). Current Genetics, 63 (1):117-129. doi: 10.1007/s00294-016-0612-4 URL
[15]	Seale M, Bennett T, Leyser O. 2017. BRC1 expression regulates bud activation potential but is not necessary or sufficient for bud growth inhibition in Arabidopsis. Development, 144 (9):1661-1673.
[16]	Shen J J, Zhang Y Q, Ge D F, Wang Z Y, Song W Y, Gu R, Che G, Cheng Z H, Liu R Y, Zhang X L. 2019. CsBRC1inhibits axillary bud outgrowth by directly repressing the auxin efflux carrier CsPIN3 in cucumber. Proceedings of the National Academy of Sciences, 116 (34):17105-17114.
[17]	Wang N, Yang H Z, Yin Z Y, Liu W T, Sun L Y, Wu Y F. 2018. Phytoplasma effector SWP 1 induces witches’ broom symptom by destabilizing the TCP transcription factor BRANCHED1. Molecular Plant Pathology, 19 (12):2623-2634. doi: 10.1111/mpp.12733 pmid: 30047227
[18]	Waters M T, Gutjahr C, Bennett T, Nelson D C. 2017. Strigolactone signaling and evolution. Annual Review of Plant Biology, 68:291-322. doi: 10.1146/annurev-arplant-042916-040925 pmid: 28125281
[19]	Wei Wei. 2013. Agrobacterium-mediated transformation of Chinese jujube with TaPT2 gene[Ph. D. Dissertation]. Baoding:Hebei Agricultural University. (in Chinese)
	魏薇. 2013. 枣外源小麦磷转运蛋白基因(TaPT2)遗传转化研究[博士论文]. 保定: 河北农业大学.
[20]	Xie X B, Li S, Zhang R F, Zhao J, Chen Y C, Zhao Q, Yao Y X, You C X, Zhang X S, Hao Y J. 2012. The bHLH transcription factor MdbHLH 3 promotes anthocyanin accumulation and fruit colouration in response to low temperature in apples. Plant,Cell & Environment, 35 (11):1884-1897.
[21]	Zhao Wei, Peng Jian-ying. 2007. Advances in tissue cultur e of Chinese jujube. Biotechnology Bulletin,(3):57-62. (in Chinese)
	赵薇, 彭建营. 2007. 枣树组织培养研究进展. 生物技术通报,(3):57-62.

引物	上游引物（5′-3′）	下游引物（5′-3′）
Primer	Forward primer	Reverse primer
qRT-ZjYUCCA2	TGGAGGAGACAGAGTACGTTAG	GGATAACAGGGCCACAGAAA
qRT-ZjYYUCCA4	GAGAATGCAGAGCCCGTAATA	CCCGACGACCAGAACTTTAT
qRT-ZjYYUCCA6	GCAGGAGTTTAGTGGGAAGAG	CACAAGAGATGGCCTAGCATTA
qRT-ZjYYUCCA7	CAGCTACTTAGAATCATATGCCAAAC	CCTCTCCGGGATTCGAGCT
qRT-ZjYYUCCA10-1	GGGAGAGAAAGGGCTGTATTG	ACCGATTGAGCAATGTGATTTG
qRT-ZjYYUCCA10-2	GATTGCTGTGCTTCGCTTTG	CGGTGTGGTAGATGTGGTAAAG
qRT-ZjYYUCCA10-3	GGAAACCACCGACCCTTATATT	ACTCCATCCCAGATCTATACCC
qRT-ZjYYUCCA10-4	ACGATTACGCTTCCCACTTC	CCTCGCTATCACCTTCCATTT
qRT-ZjYYUCCA11	GACCAACCACAAAGCAAAGAC	AGCCGGATGTCAGAAGAAAC

引物	上游引物（5′-3′）	下游引物（5′-3′）
Primer	Forward primer	Reverse primer
qRT-ZjYUCCA2	TGGAGGAGACAGAGTACGTTAG	GGATAACAGGGCCACAGAAA
qRT-ZjYYUCCA4	GAGAATGCAGAGCCCGTAATA	CCCGACGACCAGAACTTTAT
qRT-ZjYYUCCA6	GCAGGAGTTTAGTGGGAAGAG	CACAAGAGATGGCCTAGCATTA
qRT-ZjYYUCCA7	CAGCTACTTAGAATCATATGCCAAAC	CCTCTCCGGGATTCGAGCT
qRT-ZjYYUCCA10-1	GGGAGAGAAAGGGCTGTATTG	ACCGATTGAGCAATGTGATTTG
qRT-ZjYYUCCA10-2	GATTGCTGTGCTTCGCTTTG	CGGTGTGGTAGATGTGGTAAAG
qRT-ZjYYUCCA10-3	GGAAACCACCGACCCTTATATT	ACTCCATCCCAGATCTATACCC
qRT-ZjYYUCCA10-4	ACGATTACGCTTCCCACTTC	CCTCGCTATCACCTTCCATTT
qRT-ZjYYUCCA11	GACCAACCACAAAGCAAAGAC	AGCCGGATGTCAGAAGAAAC

2,4-D/（mg · L^-1）	TDZ/（mg · L^-1）	接种叶片数 Number of inoculated leaves	诱导愈伤数 Number of induced callus	愈伤组织诱导率/% Induction rate of callus
0.5	0.2	50	0 d	0 d
0.5	0.4	50	25.00 ± 0.93 b	50.33 ± 1.54 b
1.0	0.2	50	14.00 ± 0.61 c	27.09 ± 1.01 c
1.0	0.4	50	26.00 ± 1.80 b	51.28 ± 2.99 b
2.0	0.2	50	16.00 ± 3.05 c	31.31 ± 5.07 c
2.0	0.4	50	36.00 ± 2.21 a	70.22 ± 3.67 a

2,4-D/（mg · L^-1）	TDZ/（mg · L^-1）	接种叶片数 Number of inoculated leaves	诱导愈伤数 Number of induced callus	愈伤组织诱导率/% Induction rate of callus
0.5	0.2	50	0 d	0 d
0.5	0.4	50	25.00 ± 0.93 b	50.33 ± 1.54 b
1.0	0.2	50	14.00 ± 0.61 c	27.09 ± 1.01 c
1.0	0.4	50	26.00 ± 1.80 b	51.28 ± 2.99 b
2.0	0.2	50	16.00 ± 3.05 c	31.31 ± 5.07 c
2.0	0.4	50	36.00 ± 2.21 a	70.22 ± 3.67 a

外植体 Explant	接种外植体数 Number of explants	诱导的愈伤数 Number of induced callus	愈伤组织诱导率/% Induction rate of callus
茎段 Stem segments	50	2.00 ± 0.85 d	4.03 ± 1.70 d
叶片 Leaves	50	42.00 ± 1.14 b	49.31 ± 2.47 c
子叶 Cotyledons	50	49.67 ± 0.47 a	99.33 ± 0.94 a
胚轴 Hypocotyls	50	25.00 ± 1.24 c	81.30 ± 2.28 b