|
|
|
| Construction of Sesuvium portulacastrum VIGS System Based on PDS Gene Family |
| MA-Wei1,3,*, WANG Xi-Hao1,*, ZHANG Xue-Yan1,2,** |
1 College of Life Sciences/Ministry of Education Key Laboratory for Ecology of Tropical Islands, Hainan Normal University, Haikou 571158, China;
2 Hainan Dongzhaigang, Mangrove Ecosystem, Provincial Observation and Research Station, Haikou 570105, China;
3 Hainan Haikou Changliu Middle School, Haikou 570312, China |
|
|
|
|
Abstract Sesuvium portulacastrum is a succulent saline plant, which is extremely tolerant to high salt stress. Virus-induced gene silencing (VIGS) is an important research tool to characterise the gene function of plants. In order to establish an efficient VIGS system in the important salt-tolerant plant S. portulacastrum, phytoene desaturase (PDS) gene was selected as a marker gene using S. portulacastrum of Sanya as the material. According to the transcriptome data, 5 PDS family members of S. portulacastrum were identified by local Blast and other biosignature means, and the common sequences of the 5 members were obtained by PCR, with a length of 431 bp.Meanwhile, SpTRV2-PDS silencing expression vector was constructed, which was transformed by Agrobacterium tumefaciens GV3101 and then infiltrated into the plants by injection. The phenotype was observed, and the changes of carotenoid content and PDS gene expression were measured. The results showed that after infection, the plants showed slow growth and gradual yellowing of leaves, and the leaves showed albino phenomenon after 7 d, and the carotenoid content and the relative expression of PDS in the experimental group continued to exist, indicating that the VIGS system of S. portulacastrum was successfully constructed, indicating that the VIGS system could effectively silence the expression of PDS gene in S. portulacastrum. This study provides a technical means for the identification of the function of salt tolerance genes in S. portulacastrum.
|
|
Received: 12 June 2023
|
|
|
|
Corresponding Authors:
**zhangxueyan_caas@126.com
|
| About author:: * These authors contributed equally to this work |
|
|
|
[1] 陈段芬, 彭镇华, 高志民. 2008. 中国水仙八氢番茄红素脱氢酶基因(PDS)的克隆及表达分析[J]. 分子植物育种, 6(3): 574-578.
(Chen D F, Peng Z H, Gao Z M.2008. Cloning and expression analysis of PDS gene in Narcissus tazetta var. chinensis[J].Molecular Plant Breeding, 6(3): 574-578.)
[2] 郭晔, 万东艳, 柴壮壮, 等. 2019. 利用CRISPR/Cas9敲除葡萄VviPDS1基因的研究[J]. 园艺学报, 46(04): 623-634.
(Guo Y, Wan D Y, Chai Z Z, et al.2019. Knock-out analysis of VviPDS1 gene using CRISPR/Cas9 in grape vine[J]. Acta Horticulturae Sinica, 46(04): 623-634.)
[3] 李娜娜, 邵文韵, 刘畅等. 2014. 茶树八氢番茄红素脱氢酶cDNA全长克隆与表达分析[J]. 茶叶, 40(02): 69-74.
(Li N N, Shao W Y, Liu C, et al.2014. Cloning of a full-length cDNA of phytoene desaturase gene in tea plant (Camellia Sinensis) and its expression[J]. Journal of Tea, 40(02): 69-74.)
[4] 欧阳蒲月, 沈笑飞, 曾少华等. 2013. 广藿香八氢番茄红素脱氢酶PcPDS1基因克隆和序列分析[J]. 中草药, 44(17): 2446-2452.
(Ouyang P Y, Shen X F, Zeng S H, et al.2013. Cloning and sequence analysis of phytoene desaturase gene in Pogostmen cablin[J]. Chinese Traditional and Herbal Drugs, 44(17): 2446-2452.)
[5] 孙威, 许奕, 许桂莺等. 2015. 病毒诱导的基因沉默及其在植物研究中的应用[J]. 生物技术通报, 31(10): 105-110.
(Sun W, Xu Y, Xu G Y, et al.2015. Virus-induced gene silencing and lts application in plant research[J]. Biotechnology Bulletin, 31(10): 105-110.)
[6] 徐大伟, 张雨良, 檀根甲等. 2011. 棉花八氢番茄红素脱氢酶GhPDS1基因的克隆与表达谱分析[J]. 棉花学报, 23(3): 200-204.
(Xu D W, Zhang Y L, Tan G J.2011. Molecular cloning and expression analysis of GhPDS1 gene in Gossypium hirsutum[J]. Cotton Science, 23(3): 200-204.)
[7] 薛欢. 2019. 不同光强下金银忍冬幼苗生长及光合生理特性[D]. 硕士学位论文, 北华大学, 导师: 杜凤国. pp. 9-10.
(Xue H.2019. Growth and photosynthetic physiological characteristics of Lonicera maackii Maxim. seedlings under different light intensities[D]. Thesis for M.S., BeiHua University, Supervisor: Du F G,pp. 9-10.)
[8] 姚丹青, 张微微, 原丽华等. 2009. VIGS: 植物功能基因组学研究的革命[J]. 分子植物育种, 7(01): 155-161.
(Yao D Q, Zhang W W, Yuan L H, et al.2009. VIGS: The revolution of plant function genomics research[J]. Molecular Plant Breeding, 7(01): 155-161.)
[9] 于沐, 文艺, 赵辉等. 2015. 芝麻八氢番茄红素脱氢酶基因SiPDS的克隆与序列分析[J]. 分子植物育种, 13(03): 589-594.
(Yu M, Wen Y, Zhao H, et al.2015. Cloning and sequence analysis of SiPDS gene in Sesame indicum L.[J]. Molecular Plant Breeding, 13(03): 589-594.)
[10] 曾碧健, 窦碧霞, 黎祖福等. 2017. 海洋盐生植物海马齿(Sesuvium portulacastrum)对环境盐度胁迫的耐受性及营养价值综合评价[J]. 海洋与湖沼, 48(3): 568-575.
(Zeng B J, Dou B X, Li Z F, et al.2017. Salt tolerance of environmental salinity stress and comprehensive evaluation of nutritional value of Sesuvium portulacastrum. an important halophyte[J]. Oceanologia et Limnologia Sinica, 48(3): 568-575.)
[11] 赵军林. 2017. 甜瓜果实PDS基因克隆及β-胡萝卜素积累分子机理研究[D]. 硕士学位论文, 山东农业大学, 导师: 王秀峰. pp. 31-34.
(Zhao J L.2017. Cloning of melon's PDS gene and study of B-carotenoid accumulation molecular mechanism in muskmelon[D]. Thesis for M.S., Shandong Agricultural University, Supervisor: Wang X F. pp. 31-34.)
[12] 赵新伟. 2010. 番木瓜类胡萝卜素生物合成途径部分酶基因cDNA的克隆与分析[D]. 硕士学位论文, 福建农林大学, 导师: 陈晓静. pp. 15-29.
(Zhao X W.2010. Cloning and analysis of partial enzyme members cDNA in papaya (Caricapapaya L.) of carotenoid biosynthetic pathway[D]. Thesis for M.S., Fujian Agriculture and Forestry University, Supervisor: Chen X J. pp. 15-29.)
[13] Baulcombe D C.1999. Fast forward genetics based on virus-induced gene silencing[J]. Current Opinion in Plant Biology, 2(2): 109-113.
[14] Bilichak A, Kovalchuk I., 2017. Increasing a stable transformation efficiency of Arabidopsis by manipulating the endogenous gene expression using virus-induced gene silencing[J]. Methods in Molecular Biology, 1456: 225-236.
[15] Broderick S R, Chapin L J, Jones M L, 2020. Virus-induced gene silencing for functional analysis of flower traits in Petunia[J]. Methods in Molecular Biology, 2172: 199-222.
[16] Dommes A B, Gross T, Herbert D B, et al.2019. Virus-induced gene silencing: Empowering genetics in non-model organisms[J]. Journal of experimental botany, 70(3): 757-770.
[17] Fofana I B, Sangare A, Coller R, et a1.2004. A geminivirus-induced gene silencing system for gene function validation in cassara[J]. Plant Molecular Biology, 56(4): 613-624.
[18] Kobayashi H, Tomari Y.2016. RISC assembly: Coordination between small RNAs and Argonaute proteins[J]. Biochim Biophys Acta, 1859(1): 71-81.
[19] Kumagai M H, Donson J, Della-Cioppa G, et al.1995. Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA[J]. Proceedings of the National Academy of Sciences of the USA, 92(5): 1679-1683.
[20] Lentz E M, Kuon J E, Alder A, et al.2018. Cassava geminivirus agroclones for virus-induced gene silencing in cassava leaves and roots[J]. Plant Methods, 14: 73.
[21] Li C, Yamagishi N, Yoshikawa N.2019. Rna silencing-mediated Apple latent spherical virus vaccine in plants[J]. Methods in Molecular Biology, 2028: 273-288.
[22] Pandey P, Mysore K S, SenthilKumar M.2022. Recent advances in plant gene silencing methods[J]. Methods in Molecular Biology (Clifton,N.J.). 2408: 21-22.
[23] Qin C, Li B, Fan Y, et al.2017. Roles of dicer-like proteins 2 and 4 in intra- and intercellular antiviral silencing[J]. Plant Physiology, 174(2): 1067-1081.
[24] Szittya G, Burgyán J.2013. RNA interference-mediated intrinsic antiviral immunity in plants[J]. Current Topics in Microbiology and Immunology, 371: 153-181.
[25] Yamagishi N, Yoshikawa N.2010. Virus-induced gene silencing as a tool for analysis of gene functions in plants[J]. Uirusu, 60(2): 155-162. |
|
|
|
