Effects of Silencing Lcyb and BZR1 Genes on Carotenoid Accumulation in Leaves of Pepper (Capsicum annuum)
LI Jie, LUO Jiang-Hong, YANG Ping*
Key Laboratory for Research and Utilization of Characteristic Biological Resources in Southern Yunnan/College of Life Science and Technology, Honghe University, Mengzi 661100, China
Abstract：Carotenoids, as important members of photosynthetic system and antioxidant system in plants, play important roles in plant growth and responding to stress. In order to study the effects of lycopene β-cyclase (Lcyb) and brassinazole-resistant 1 (BZR1) genes on the regulation of carotenoid biosynthesis, the virus-induced gene silencing (VIGS) system of Lcyb and BZR1 gene mediated by Tobacco rattle virus (TRV) was constructed in pepper (Capsicum annuum 'Shan Zao hong'). The gene expression of Lcyb and BZR1 and seedlings of RNAi lines were obtained by vein injection. The uninfected pepper plants were used as the normal control, the infected plants with TRV2-PDS gene were used as the positive control, and the infected plants with empty TRV2 vector were used as the negative control. qRT-PCR was used to detect the Lcyb and BZR1 genes expression of silenced pepper, and the accumulation of carotenoids in silenced pepper leaves was determined. Results showed that TRV2-PDS leaves appeared bleaching phenomenon after 21 d, and Lcyb and BZR1 gene expression were significantly lower (P<0.05), and carotenoid content of 2 kinds of silenced pepper leaf was significantly lower than that of normal control plants. It is indicated that Lcyb and BZR1 genes were involved in synthesis of carotenoids in pepper leaf. The study of TRV-mediated gene-silencing could be used for subsequent functional gene identification of pepper.
李杰, 罗江宏, 杨萍. 沉默辣椒Lcyb和BZR1基因对其叶片类胡萝卜素积累的影响[J]. 农业生物技术学报, 2021, 29(1): 47-57.
LI Jie, LUO Jiang-Hong, YANG Ping. Effects of Silencing Lcyb and BZR1 Genes on Carotenoid Accumulation in Leaves of Pepper (Capsicum annuum). 农业生物技术学报, 2021, 29(1): 47-57.
[1] 毕玉平, 单蕾, 王兴军, 等. 1999. 双抗TMV+CMV辣椒转基因工程植株的再生及抗病毒鉴定[J]. 华北农学报, 14(3): 103-108. (Bi Y P, Shan L, Wang X J, et al.1999. Regeneration of transgenic pepper plants resistant to TMV and CMV and its resistance assay[J]. Acta Agriculturae Boreali-Sinica, 14(3): 103-108.) [2] 黄季焜, 王济民, 解伟, 等. 2019. 现代农业转型发展与食物安全供求趋势研究[J]. 中国工程科学, 21(5): 1-9. (Huang J K, Wang J M, Xie W, et al.2019. Modern agricultural transformation and trend of food supply and demand in China[J]. Strategic Study of CAE, 21(5): 1-9.) [3] 李成伟, 裴冬丽. 2007. 植物病毒载体在植病互作研究中的应用[J]. 生物技术通报, 7(4): 27-30. (Li C W, Pei D L.2007. Application of plant virus vectors in plant-pathogen interaction study[J]. Biotechnology Bulletin, 7(4): 27-30.) [4] 李聪聪, 安晓晖, 张中起, 等. 2019. 玉米TRV-VIGS的优化与顶腐病抗病基因的鉴定[J]. 核农学报, 33(11): 2111-2118. (Li C C, An X H, Zhang Z Q, et al.2019. Optimization of TRV-VIGS system and identification of top rot resistance related genes in maize[J]. Journal of Nuclear Agricultural Sciences, 33(11): 2111-2118.) [5] 刘晓彬, 刘娜, 李福宽, 等. 2015. TRV介导的大豆基因瞬时沉默体系的建立[J]. 中国农业科学, 48(12): 198-205. (Liu X B, Liu N, Li F K, et al.2015. Establishment of TRV-mediated transient gene-silencing system in soybean[J]. Scientia Agricultura Sinica, 48(12): 198-205.) [6] 宋恬, 田嘉, 李鹏, 等. 2019. 扁桃AcCBF1基因VIGS载体构建与功能分析[J]. 果树学报, 36(04): 31-39. (Song T, Tian J, Li P, et al.2019. Vector construction and functional analysis of AcCBF1 using VIGS method in almond flower organs[J]. Journal of Fruit Science, 36(04): 31-39.) [7] 宋震, 李中安, 周常勇. 2014. 病毒诱导的基因沉默(VIGS)研究进展[J]. 园艺学报, 41(09): 1885-1894. (Song Z, Li Z A, Zhou C Y.2014. Research advances of virus-induced gene silencing (VIGS)[J]. Acta Horticulturae Sinica, 41(09): 1885-1894.) [8] 王曼曼, 薛舒丹, 吴廷全, 等. 2020. 光照和温度调控对番茄果实中类胡萝卜素合成的影响[J]. 分子植物育种, 18(18): 6158-6164. (Wang M M, Xue S D, Wu T Q, et al.2020.Effects of illumination and temperature regulation on synthesis of caroteno genesis in tomato (Solanum lycopersicum L.) fruit[J]. Molecular Plant Breeding, 18(18): 6158-6164.) [9] 王瑞鹏, 冯国栋, 徐焕焕, 等. 2019. TePDS1基因功能番茄遗传转化分析[J]. 安徽农业科学, 47(22): 107-110. (Wang R P, Feng G D, Xu H H, et al .2019. Function analysis of TePDS1 gene by transformation in tomato[J]. Journal of Anhui Agricutural Sciences, 47(22): 107-110.) [10] 杨迎伍, 李正国, 宋红丽, 等. 2007. VIGS技术在植物基因功能研究中的应用[J]. 植物生理学通讯, 43(02): 379-383. (Yang Y W, Li Z G, Song H L, et al.2007. Application of VIGS in plant gene function study[J]. Plant Physiology Communications, 43(02): 379-383.) [11] 张玉文. 2017. 青花菜BZR1、BES1转录因子的克隆与功能分析[D].硕士学位论文, 福建农林大学, 导师: 郑金贵. pp: 56-57. (Zhang Y W.2017. Cloning and functional analysis of transcription factors 1 and BES1 in broccoli[D]. Thesis for M.S., Fujian Agriculture and Forestry University, Supervisor: Zheng J G, pp: 56-57.) [12] 赵梦娜, 梁晶, 张克中, 等. 2019. 利用基因沉默技术验证东方百合'Justina'番茄红素β-环化酶基因的功能[J]. 北京农学院学报, 34(03): 81-86. (Zhao M N, Liang J, Zhang K Z, et al.2019. Function validation of LoLcyB gene in Lilium orientalis 'Justina' by VIGS system[J]. Journal of South Chine Normal (Natural Science Edition), 34(03): 81-86.) [13] Ali N, Ansar H, Muhammad A, et al.2019. A novel MYB transcription factor CaPHL8 provide clues about evolution of pepper immunity against soil borne pathogen[J]. Microbial Pathogenesis, 137(11): 103758. [14] Becker A, Lange M.2010. VIGS-Genomics goes functional[J]. Trends in Plant Science, 15(1): 1-4. [15] Burch-Smith T M, Anderson J C, Martin G B, et al.2004. Applications and advantages of virus induced gene silencing for gene function studies in plants[J]. Plant Journal, 39(5): 734-746. [16] del Rosario Abraham-Juárez M, Del Carmen RochaGranados M, López M G, et al.2008. Virus-induced silencing of Comt, pAmt and Kas genes results in a reduction of capsaicinoid accumulation in chili pepper fruits[J]. Planta, 227(3): 681-695. [17] Ekengren S K, Liu Y, Schiff M, et al.2003. Two MAPK cas-cades, NPR1, and TGA transcription factors play a role in Pto-mediated disease resistance in tomato[J]. Plant Journal, 36(6): 905-917. [18] Fu D Q, Zhu B Z, Zhu H L, et al.2006. Enhancement of virus induced gene silencing in tomato by low temperature and low humidity[J]. Molecules and Cells, 21(1): 153-160. [19] Hartl M, Merker H, Schmidt D D, et al.2008. Optimized virus-induced gene silencing in Solanum nigrum reveals the defensive function of leucine aminopeptidase against herbivores and the shortcomings of empty vector controls[J]. New Phytologist, 179(2): 356-365. [20] Huh S U, Kim K J, Paek K H.2012. Capsicum annuum basic transcription factor 3 (CaBtf3) regulates transcription of pathogenesis-related genes during hypersensitive response upon Tobacco mosaic virus infection[J]. Biochemical and Biophysical Research Communications, 417(2): 910-917. [21] Igarashi A, Yamagata K, Sugai T, et al.2009. Apple latent spherical virus vectors for reliable and effective virus-induced gene silencing amonga broad range of plants including tobacco, tomato, Arabidopsis thaliana, cucurbits, and legumes[J]. Virology, 386(2): 407-416. [22] Jacob S S, Vanitharani R, Karthikeyan A S, et al.2003. Mungbean yellow mosaic virus-Vi agromfection by codelivery of DNA A and DNA B from one Agrobacterium strain[J]. Plant Disease, 87(03): 247-251. [23] Kjemtrup S, Sampson K S, Peele C G, et al.1998. Gene silencing from plant DNA carried by a Geminivirus[J]. The Plant Journal, 14(1): 91-100. [24] Kumagai M H, Dodson 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. [25] Lim S, Baek W, Lee S C.2014. Identification and functional roles of CaDIN1 in abscisic acid signaling and drought sensitivity[J]. Plant Molecular Biology, 86(4-5): 513-525. [26] Liu E, Page J E.2008. Optimized cDNA libraries for virus-induced gene silencing (VIGS) using Tobacco rattle virus[J]. Plant Methods, 4: 5. [27] Liu L J, Jia C G, Zhang M, et al.2014. Ectopic expression of a BZR1-1D transcription factor in brassinosteroid signalling enhances carotenoid accumulation and fruit quality attributes in tomato[J]. Plant Biotechnology Journal, 12(01): 105-115. [28] Liu Y L, Schiff M, Dinesh-Kumar S P.2002. Virus-induced gene silencing in tomato[J]. The Plant Journal, 31(6): 777-786. [29] Naim F, Dugdale B, Kleidon J, et al.2018. Gene editing the phytoene desaturase alleles of Cavendish banana using CRISPR/Cas9[J]. Transgenic Research, 27(5): 451-460. [30] Nethra P, Nataraja K N, Rama N, et al.2006. Standardization of environment conditions for induction and retention of post-transcriptional gene silencing using Tobacco rattle virus vector[J]. Current Science, 90(3): 431-435. [31] Qin C, Yu C S, Shen Y, et al.2014. Whole-genome sequencing of cultivated and wild peppers provides insights into Capsicum domestication and specialization[J]. Proceedings of the National Academy of Sciences of the USA, 111(14): 5135-5140. [32] Ratcliff F, Martin-Hernandez A M, Baulcombe D C.2001. Tobacco rattle virus as a vector for analysis of gene function by silencing[J]. The Plant Journal, 25(2): 237-245. [33] Rodrigo M J, Marcos J F, Zacarias L.2004. Biochemical and molecular analysis of carotenoid biosynthesis in flavedo of orange (Citrus sinensis L.) during fruit development and maturation[J]. Journal of Agricultural and Food Chemistry, 52(22): 6724-6731. [34] Rodriguez-Uribe L, Guzman I, Rajapakse W, et al.2012. Carotenoid accumulation in orange pigmented Capsicum annuum fruit, regulated at multiple levels[J]. Journal of Experimental Botany, 63(1): 517-526. [35] Ruiz M T, Voinnet O, Baulcombe D C.1998. Initiation and maintenance of virus-induced gene silencing[J]. The Plant Cell, 10(6): 937-946. [36] Sun Y, Fan X Y, Cao D M, et al.2010. Integration of brassinosteroid signal transduction with the transcription network for plant growth regulation in Arabidopsis[J]. Developmental Cell, 19(05): 765-777. [37] Turnage M A, Muangsan N, Peele C G, et al.2002. Germinivirus-based vectors for gene silencing in Arabidopsis[J]. The Plant Journal, 30(1): 107-114. [38] Welsch R, Beyer P, Hugueney P, et al.2000. Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis[J]. Planta, 211(6): 846-854. [39] Zheng S J, Snoeren T A L, Hogewoning S W, et al.2010. Disruption of plant carotenoid biosynthesis through virus-induced gene silencing affects oviposition behaviour of the buterfly Pieris rapae[J]. New Phytologist, 186(03): 733-745.
