Cloning and Identification of LHY Gene in Chinese Milk Vetch (Astragalus sinicus)
ZHANG Xian1, ZHUANG Li1, CAO Wei-Dong2, CAO Kai1, XU Jing1, SI Lin-Lin1, WANG Jian-Hong1,*
1 Institute of Environment, Resource, Soil & Fertilizer, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China; 2 Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081, China
Abstract：Astragalus sinicus has been identified as one of the most important green manure for paddy fields in the south of China. It is vital to the sustainable utilization of soil resources. Based on different farming systems, selecting flowering varieties is an important breeding target of A. sinicus. Flowering time is regulated by the circadian clock in response to the external environment. Late elongated hypocoty l (LHY) is a key biorhythm clock gene. In the present study, in order to explore the function of the LHY gene in A. sinicus, the Illumina and rapid-amplification of cDNA ends (RACE) technology were performed to obtain the sequence of LHY in A. sinicus; the expression profiles of the LHY gene in tissues of A. sinicus were subsequently analyzed by qRT-PCR; the function of LHY gene of A. sinicus is verified by the transformation of LHY gene into Arabidopsis. The full-length cDNA of the LHY gene (GenBank No. OP455117) was isolated in A. sinicus, which was 2 865 bp in length and containing the ORF of 2 259 bp, encoding 752 amino acid polypeptide. The encoded amino acid sequence is highly homologous to the LHY proteins of Medicago truncatula, Cicer arietinum, and Glycine soja with a more than 75% similarity. The qRT-PCR analysis showed that the LHY gene had the highest expression accumulation in leaves, flowers, and flower buds of Astragalus sinicus. The LHY-overexpression transgenic Arabidopsis lines showed a significant late flowering phenotype. The bolting days and flowering days of transgenic Arabidopsis lines were 18.36 and 20.72 d later than that of the wild-type respectively. This study provides important implications for genetic modification of flowering in A. sinicus.
[1] 曹卫东, 黄鸿翔. 2009. 关于我国恢复和发展绿肥若干问题的思考[J]. 中国土壤与肥料, (12): 1-3. (Cao W D, Huang H X. 2009. Ideas on restoration and development of green manures in China[J]. Soil and Fertilizer Sciences in China, (12): 1-3.) [2] 陈默君, 贾慎修. 2002. 中国饲用植物[M]. 北京: 中国农业出版社, pp. 413-415. (Chen M J, Jia S X.2002. Forage plant of China[M]. Agricultural Press, Beijing, China, pp. 413-415.) [3] 林多胡, 顾荣申. 2000. 中国紫云英[M]. 福州:福建科学技术出版社, pp. 69-83. (Lin D F, Gu R S.2000. Astragalus sinicus L. of China[M]. Fujian Science and Technology Publishing Press, Fuzhou, China, pp. 69-83.) [4] 林新坚, 曹卫东, 吴一群, 等. 2011. 紫云英研究进展[J]. 草业科学, 28(1): 135-140. (Lin X J, Cao W D, Wu Y Q, et al.2011. Advances in the research of Astragalus sinicus[J]. Pratacultural Science, 28(1): 135-140.) [5] 潘福霞, 李小坤, 鲁剑巍, 等. 2012. 不同播期对紫云英生长及物质养分积累的影响[J]. 土壤, 44(1): 67-72. (Pan F X, Li X K, Lu J W, et al.2012. Effects of sowing date on Chinese milk vetch growth and nutrient accumulation[J]. Soils, 44(1): 67-72.) [6] 张贤, 王建红, 曹凯, 等. 2018. 紫云英LEAFY基因的克隆与鉴定[J]. 农业生物技术学报, 26(8): 1328-1341. (Zhang X, Wang J H, Cao K, et al.2018. Cloning and identification of LEAFY gene in Chinese milk vetch (Astragalus sinicus)[J]. Journal of Agricultural Biotechnology, 26(8): 1328-1341.) [7] Alabadi D, Yanovsky M J, Mas P, et al.2002. Critical role for CCA1 and LHY in maintaining circadian rhythmicity in Arabidopsis[J]. Current Biology, 12: 757-761. [8] Eckardt N A.2005. A time to grow, a time to flower[J]. Plant Cell, 17(10): 2615-2617. [9] Jonas A H D, Wolf B F.2013. Jack of all trades, master of flowering[J]. Science, 339(6120): 659-660. [10] Kim M Y, Shin J H, Kang Y J, et al.2012. Divergence of flowering genes in soybean[J]. Journal of Biosciences, 37(5):857-870. [11] Komeda Y.2004. Genetic regulation of time to flower in Arabidopsis thaliana[J]. Annual Review of Plant Biology, 55(1): 521-535. [12] Li C, Liu X J, Yan Y, et al.2022. OsLHY is involved in regulating flowering through the Hd1- and Ehd1- mediated pathways in rice (Oryza sativa L.)[J]. Plant Science, 315: 111145. [13] Lu S X, Knowles S M, Andronis C, et al.2009. Circadian clock associated1 and late elongated hypocotyl function synergistically in the circadian clock of Arabidopsis[J]. Plant Physiology, 150: 834-843. [14] Mizoguchi T, Wheatley K, Hanzawa Y, et al.2002. LHY and CCA1 are partially redundant genes required to maintain circadian rhythms in Arabidopsis[J]. Developmental Cell, 2: 629-641. [15] Park M J, Kwon Y J, Gil K E, et al.2016. Late elongated hypocotyl regulates photoperiodic flowering via the circadian clock in Arabidopsis[J]. BMC Plant Biology, 16: 114. [16] Schaffer R, Ramsay N, Samach A, et al.1998. The late elongated hypocotyl mutation of Arabidopsis disrupts circadian rhythms and the photoperiodic control of flowering[J]. Cell, 93(7): 1219-1229. [17] Shim J S, Kubota A, Imaizumi T.2017. Circadian clock and photoperiodic flowering in Arabidopsis: CONSTANS is a hub for signal integration[J]. Plant Physiology, 173(1): 5-15. [18] Simpson G G, Dean C.2002. Arabidopsis, the Rosetta stone of flowering time[J]. Science, 296(5566): 285-289 [19] Song H R, Noh Y S.2007. Plants measure the time[J]. Journal of Plant Biology, 50(3): 257-265. [20] Song Y H, Shim J S, Kinmonth S H A, et al.2015. Photoperiodic flowering: Time measurement mechanisms in leaves[J]. Annual Review of Plant Biology, 66: 441-464. [21] Wahl V, Jathish P, Armin S, et al.2013. Regulation of flowering by trehalose-6-phosphate signaling in Arabidopsis thaliana[J]. Science, 339(6120): 704-707. [22] Yang Z, Xu M, Zheng S, et al.2012. Effects of long-term winter planted green manure on physical properties of reddish paddy soil under a double-rice cropping system[J]. Journal of Integrative Agriculture, 11(4): 655-664. [23] Zhang C, Cheng Q, Li H, et al.2020. DhLHY, a circadian expressed gene of doritaenopsis hybrid, promotes floral transition in low temperature, but postpones flowering in overexpressed transgenic Arabidopsis[J]. Tropical Plant Biology, 13: 162-171.
