Response of Receptor-Like Protein Kinase Gene SiRLK35 of Foxtail Millet to Salt in Heterologous Transgenic Rice
LI XiaoBo1,2, LI Zhen2, DAI ShaoJun3, PAN JiaoWen2, WANG QingGuo2, GUAN YanAn4,5, DING GuoHua,1, LIU Wei,2,51 School of Life Sciences and Technology, Harbin Normal University, Harbin 150025 2 Biotechnology Research Center, Shandong Academy of Agricultural Sciences/Shandong Provincial Key Laboratory of Crop Genetic Improvement, Ecology and Physiology, Jinan 250100 3 College of Life and Environmental Sciences, Shanghai Normal University/Development Center of Plant Germplasm Resources, Shanghai 200234 4 Crop Research Institute, Shandong Academy of Agricultural Sciences/Shandong Engineering Laboratory for Featured Crops, Jinan 250100 5 College of Life Sciences, Shandong Normal University, Jinan 250014
Abstract 【Objective】Salt stress affects the crop yield and quality, while the crop salt tolerance is regulated by specific genes. Using heterogeneous over-expressed rice lines (OE-1, OE-2, and OE-3) of foxtail millet receptor-like protein kinase gene SiRLK35, the possible mechanisms of SiRLK35 under salinity will be dissected on phenotypic, physiological and molecular levels. 【Method】 The expressions of SiRLK35 in OE-1, OE-2, and OE-3 were analyzed by qRT-PCR. The phenotypes of three-leaf stage seedlings treated with 0, 150 and 200 mmol·L -1 NaCl were observed, and the lengths of seedlings and roots were measured after treated with 150 mmol·L -1 NaCl for 3 days. The dry weights, death rate and dead leaf rate of four-leaf stage seedlings that treated with 150 mmol·L -1 NaCl for 14 days were also measured. OE-1with the highest SiRLK35 expression was further used for DAB and NBT dyeing analysis. The activities of partial antioxidases, and expression patterns of marker genes were detected. 【Result】 There was the highest SiRLK35 expression level in OE-1. The growth of control and OE seedlings were all inhibited under 150 mmol·L -1 NaCl. The decreased levels of dry weight, the death rate and dead leaf rate of OE rice were all lower than those of the control, together with the less accumulation of O 2- and H2O2, and the higher activities of antioxidases under salinity. Partial of salt responsive genes were up-regulated in SiRLK35 OE lines, especially the OsLEA3 was induced about 1.9 times higher after treated with NaCl for 24 h. 【Conclusion】 SiRLK35 OE rice lines have tolerance under salinity, and the gene SiRLK35 of foxtail millet could participate in salt response by regulation the activities of antioxidases and related signal pathways. Keywords:foxtail millet (Setaria italica L.);SiRLK35;salt tolerance;antioxidase;salt responsive genes
PDF (1208KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 李小波, 李臻, 戴绍军, 潘教文, 王庆国, 管延安, 丁国华, 刘炜. 谷子类受体蛋白激酶基因SiRLK35在水稻中对盐胁迫的响应[J]. 中国农业科学, 2019, 52(22): 3976-3986 doi:10.3864/j.issn.0578-1752.2019.22.004 LI XiaoBo, LI Zhen, DAI ShaoJun, PAN JiaoWen, WANG QingGuo, GUAN YanAn, DING GuoHua, LIU Wei. Response of Receptor-Like Protein Kinase Gene SiRLK35 of Foxtail Millet to Salt in Heterologous Transgenic Rice[J]. Scientia Acricultura Sinica, 2019, 52(22): 3976-3986 doi:10.3864/j.issn.0578-1752.2019.22.004
0 引言
【研究意义】盐胁迫是一种典型的非生物胁迫,是影响农作物生长发育和产量的主要因素之一[1]。盐胁迫能够打乱植物体内正常的离子分布和动态平衡,破坏细胞内代谢过程,导致细胞内活性氧积累、细胞膜过氧化、生物大分子破坏,最终抑制植物的生长发育,甚至引起植物死亡[2]。据统计,世界盐碱地面积已达9.54×108 hm2,占可耕地面积的10%。中国拥有盐碱地9 913×104 hm2,占耕地面积的20%[3]。盐碱地作为中国重要的后备土地资源,因其土壤质量差、生产效率低而大面积荒芜,导致这些地区农业生产不足,严重制约其区域经济发展[4]。因此,研究农作物耐盐机制、培育抗逆耐盐新品种,是农业研究者们的重大责任。谷子(Setaria italica L.)作为中国特色杂粮作物,具有C4光合途径、耐贫瘠、抗逆性强等特点,近年来已逐渐成为研究C4和抗旱抗逆的模式作物[5]。鉴定谷子抗逆基因及其功能对于作物遗传改良及抗逆品种培育具有重要意义。【前人研究进展】类受体蛋白激酶(lectin receptor-like protein kinase,LecRLKs)是植物中最大的基因家族,在调控植物的生长发育、抗逆及抗病等方面发挥重要作用[6]。其中,凝集素类受体蛋白激酶(LecRLKs)参与盐胁迫响应的报道较多[7]。位于质膜系统中的豌豆凝集素类受体蛋白激酶基因PsLecRLK在盐胁迫条件下表达明显上调,其可通过增强下游水通道蛋白基因表达来提高过表达株系的吸水能力,从而减少ROS的积累,提高过表达株系的抗盐性[8]。水稻基因SIT1是一种主要在根表皮细胞中表达并介导盐敏感的LecRLK,参与盐胁迫信号的感知和传导。盐胁迫激活SIT1表达,通过磷酸化MAPK3/ MAPK6,促进乙烯的合成并增加了活性氧的积累,进而导致植株在盐胁迫下生长受抑制甚至死亡[9]。通过抑制SIT1的表达和降低SIT1的活性,可明显提高植物的耐盐性。除LecRLKs外,其他类型的RLK基因也参与盐胁迫响应。PnRLK-1是南极苔藓中的一种细胞质型类受体蛋白激酶,在盐处理下,PnRLK-1能够上调一系列ROS清除基因如AtAPX1、AtZAT10和AtCAT1的表达,从而减少ROS积累,提高植物对盐胁迫的耐受性[10];OsRMC是水稻中发现的富含半胱氨酸的类受体蛋白激酶,是水稻盐胁迫应答中的负调控因子,其转录受2个AP2/ERF转录因子OsEREBP1和OsEREBP2的负调控[11]。近年来,也有对谷子抗盐基因的研究报道,如谷子液泡H+-ATPase E亚基基因SiVHA-E可通过正向调控ABA信号途径以及减少植株体内Na+积累和水分散失,从而显著提高拟南芥耐盐性[12];黄锁等[13]发现谷子核转录因子基因SiNF-YA5能够提高拟南芥中盐胁迫应答基因NHX1和LEA7的表达,且主要通过ABA非依赖途径提高转基因植物对盐胁迫的耐性;秦玉海等[14]发现谷子SibZIP42转基因拟南芥株系在种子萌发时期耐盐性显著提高,推测SibZIP42可能通过ABA信号途径正向调控植物的耐盐性;谷子基因SiASR4在植物抵抗干旱和盐胁迫中发挥着重要作用,其表达受DREB类转录因子SiARDP调控,通过ABA信号通路参与植物对非生物胁迫的响应过程[15]。谷子SiRLK35(NCBI登录号:XM_004956247.2)是作物逆境生物学研究组前期以谷子豫谷1号为材料,通过iTRAQ技术筛选到的一个干旱响应的类受体蛋白激酶基因。该基因编码蛋白含有392个氨基酸,包含一个S-TKc保守结构域以及一个TFS2N结构域,含3个跨膜结构域,N端有信号肽。通过构建双元植物表达载体pCAMBIA1301P- SiRLK35,已获得基因过表达水稻株系,初步分析显示该基因可提高转基因水稻的抗盐性[16,17],参与植物的抗逆响应。【本研究切入点】近年来,谷子作为一种新型模式作物受到广泛关注,但有关谷子耐盐基因的鉴定及相关机制研究鲜有报道。开展该方面研究对于解析谷子抗盐、耐逆机制,开展作物耐盐品种的改良及选育具有重要意义。【拟解决的关键问题】本研究通过对盐害下谷子SiRLK35异源转化获得的过表达水稻株系表型观察及鉴定,明确SiRLK35在作物盐胁迫响应中的作用,并对其参与机制深入解析,为改良及培育作物耐盐品种提供新的思路。
取生长至三叶期各株系水稻幼苗叶片,-80℃保存备用。在DNA水平对过表达株系的转基因阳性进行鉴定。参照SiRLK35全长序列,利用Primer Premier 5设计引物,以水稻ACTIN(LOC_ Os03g50885)为内参,利用荧光定量PCR检测SiRLK35在过表达株系中的相对表达量,引物序列见表1。使用2-ΔCt法计算SiRLK35在各过表达株系中的相对表达量。
Table 1 表1 表1基因信息及特异性引物 Table 1Genes information and specific primers
***:与水稻中花11相比,OE水稻各株系中SiRLK35的相对表达量在P<0.001水平上差异显著(n=3)。下同 Fig. 2The relative expression levels of SiRLK35 in control and transgenic rice lines
***: Indicates that compared with Zhonghua11, the relative expression levels of SiRLK35 in OE rice lines were significantly different at P<0.001 (n = 3). The same as below
A: The phenotypes of control and transgenics treated with 0 , 150 and 200 mmol·L-1 NaCl for 2 days, Bar=5 cm; B:Plant height of control and transgenics treated with 0 and 150 mmol·L-1 NaCl for 3days; C: Root length of control and transgenics treated with 0 and 150 mmol·L-1 NaCl for 3days; D: Relative reduction of dry weight in the above-ground part of control and transgenics treated with 0 and 150 mmol·L-1 NaCl for 14 days; E: Relative reduction of dry weight in underground parts of control and transgenics treated with 0 and 150 mmol·L-1 NaCl for 14 days; F: Death rate of control and transgenics treated with 150 mmol·L-1 NaCl for 14 days; G: Dead leaf rate of control and transgenics treated with 150 mmol·L-1 NaCl for 14 days;* significantly different at P< 0.05 level (n = 3); ** significantly different at P< 0.01 level (n = 3). The same as below
A:150 mmol·L-1 NaCl处理3 d后对对照及OE-1叶片的DAB和NBT染色,标尺=1cm;B:150 mmol·L-1 NaCl处理后对照及OE-1的SOD活性;C:150 mmol·L-1 NaCl处理后对照及OE-1的POD活性;不同小写字母表示差异显著(P<0.05)(n=3) Fig. 4Detection of reactive oxygen and antioxidant enzyme activities of control and OE-1 after salt treatment
A: DAB and NBT staining analysis of control and OE-1 after 3 day of NaCl treatment, Bar=1cm; B: SOD activity analysis of control and OE-1 after salt treatment; C: POD activity of control and OE-1 after salt treatment; the different letters above the curve indicate significant difference (P<0.05) of different treatments(n=3)
ZHANG JF, ZHANG XD, ZHOU JX, LIU GH, LI DX . World resources of saline soil and main amelioration measures Research of Soil and Water Conservation, 2006,12(6):28-30. (in Chinese) [本文引用: 1]
LIU ZX, ZHANG HX, YANG XY . Effects of soil salinity on growth, ion relations, and compatible solute accumulation of two sumac species:Rhus glabra and Rhus trilobata. Communications in Soil Science and Plant Analysis, 2013,44(21):3187-3204. [本文引用: 1]
贾冠清, 刁现民 . 谷子(Setaria italica ( L.) P. Beauv. )作为功能基因组研究模式植物的发展现状及趋势 生命科学, 2017,29(3):292-301. [本文引用: 1]
JIA GQ, DIAO XM . Current status and perspectives of researches on foxtail millet ( Setaria italica( L.) P. Beauv.): A potential model of plant functional genomics studies. Chinese Bulletin of Life Sciences, 2017,29(3):292-301. (in Chinese) [本文引用: 1]
YE YY, DING YF, JIANGQ, WANG FJ, SUN JW, ZHUC . The role of receptor-like protein kinases (RLKs) in abiotic stress response in plants Plant Cell Report, 2017,36:235-242. [本文引用: 2]
LIM CW, YANG SH, SHIN KH, LEE SC, KIM SH . The AtLRK10L1.2, Arabidopsis, ortholog of wheat LRK10, is involved in ABA-mediated signaling and drought resistance. Plant Cell Report, 2015,34(3):447-455. [本文引用: 1]
VAIDN, PANDEYP, SRIVASTAVA VK, TUTEJAN . Pea lectin receptor-like kinase functions in salinity adaptation without yield penalty, by alleviating osmotic and ionic stresses and upregulating stress-responsive genes Plant Molecular Biology, 2015,88(1/2):1-14. [本文引用: 1]
LI CH, SUNY . The receptor-like kinase SIT1 mediates salt sensitivity by activating MAPK3/6 and regulating ethylene homeostasis in rice. The Plant Cell, 2014,26(6):2538-2553. [本文引用: 1]
ZHANG PY, ZHANG ZH, WANGJ, CONG BL, CHEN KS, LIU SH . A novel receptor-like kinase (PnRLK-1) from the Antarctic Moss Pohlia nutans enhances salt and oxidative stress tolerance Plant Molecular Biology Reporter, 2014,33:1156-1170. [本文引用: 1]
SERRA TS, FIGUEIREDO DD, CORDEIRO AM, ALMEIDA DM, LOURENCT, ABREU IA, SEBASTIANA, FERNANDESL, CONTRERAS MB, OLIVEIRA MM, SAIBO N JM . OsRMC, a negative regulator of salt stress response in rice, is regulated by two AP2/ERF transcription factors. Plant Molecular Biology, 2013,82(4/5):439-455. [本文引用: 1]
QIN YH, ZHANG XH, FENGL, LI WW, XU ZS, LI LC, ZHOU YB, MA YZ, DIAO XM, JIA GQ, CHENM, MIN DH . Response of Millet transcription factor gene SibZIP42 to high salt and ABA treatment in transgenic Arabidopsis. Scientia Agricultura Sinica, 2016,49(17):3276-3286. (in Chinese) [本文引用: 1]
LI JR . Function analysis of the foxtail millet (Setaria italica) gene, SiASR4, in response to drought and salt stresses of plants [D]. Beijing: China Agricultural University, 2018. (in Chinese) [本文引用: 1]
WANG YF, LIZ, PAN JW, LI YX, WANG QG, GUAN YA, LIUW . Cloning and functional analysis of the SiRLK35 gene in Setaria italic L. Hereditas (Beijing), 2017,39(5):413-422. (in Chinese) [本文引用: 4]
WANG YF, LIZ, PAN JW, WANG QG, LIUW . Cloning of receptor-like protein kinase gene SiRLK35 from Foxtail Millet and its prokaryotic expression. Shandong Agricultural Sciences, 2016,48(9):1-5. (in Chinese) [本文引用: 3]
KUMARD, YUSUF MA, SINGHP, SARDARM, SARIN NB . Modulation of antioxidant machinery in alpha-tocopherol-enriched transgenic Brassica juncea plants tolerant to abiotic stress conditions. Protoplasma, 2013,250(5):1079-1089. [本文引用: 1]
YUJ, CHENS, ZHAOQ, WANGT, YANGC, DIAZC, SUNG, DAIS . Physiological and proteomic analysis of salinity tolerance in Puccinellia tenuifora. Journal of Proteome Research, 2011,10:3852-3870. [本文引用: 1]
LI XB, PAN JW, DING GH, LIZ, WANG QG, LIUW . Determination of pH value in root cells of Foxtail Millet under salt stress Shandong Agricultural Sciences, 2019,51(2):40-44. (in Chinese) [本文引用: 1]
FENG GN, ZHANG XM, QIUM, LUO AL . Advance in rice quantitative trait loci Journal of Anhui Agricultural Sciences, 2004,32(6):1231-1234. (in Chinese) [本文引用: 1]
ZHENG CK, DOU YH, XIE LX, XIE XZ . Research progress on the genes related to salt tolerance in rice Molecular Plant Breeding, 2017,15(11):4411-4422. (in Chinese) [本文引用: 1]
ZHU WW, MA TY, ZHANG MJ, SHAW . Research progress of receptor-like protein kinases in plants Genomics and Applied Biology, 2018,37(1):451-458. (in Chinese) [本文引用: 1]
KUIPERD, SCHUITJ, KUIPER P JC . Actual cytokinin concentration in plant tissue as an indicator for salt resistance in cereals Plant Soil, 1990,123(2):243-250. [本文引用: 1]
LI JT, ZHAO PP, QIU ZB . Effects of exogenous H2O2 on physiological indexes of wheat seedlings under salt stress Acta Botanica Boreali-occidentalia Sinica, 2012,32(9):1796-1801. (in Chinese) [本文引用: 1]
MANIMARANP, VENKATA RS, MAZAHARM, RAGHURAMIM, POLIY, MOHANRAJ SS, BALACHANDRAN SM, KIRTI PB . Activation-tagging in indica rice identifies a novel transcription factor subunit, NF-YC13 associated with salt tolerance. Scientific Reports, 2017,24(4):1-16. [本文引用: 1]
ANOOPN, GUPTAK . Transgenic indica rice CV IR-50 over expressing vigna aconitifolia-pyrroline-5-carboxylatelate synthetase cDNA shows tolerance to high salt. Journal of Plant Biochemistry and Biotechnology, 2003,12(2):109-116. [本文引用: 1]
FUKUDAA, NAKAMURAA, HARAN, TOKIS, TANAKAY . Molecular and functional analyses of rice NHX-type Na+/H+ antiporter genes Planta, 2011,233(1):175-188. [本文引用: 1]
LIU SP, ZHENG LQ, XUE YH, ZHANGQ, WANGL, SHOU HX . Overexpression of OsVP1 and OsNHX1 increases tolerance to drought and salinity in rice. Journal of Plant Biology, 2010,53(6):444-452. [本文引用: 1]
NAKASHIMAK, TRAN LS, VAN ND, FUJITAM, MARUYANAK, TODAKAD, HAYASHIN, SHINOZAKIK, YAM SK . Functional analysis of a NAC type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. The Plant Journal, 2007,51(4):617-630. [本文引用: 1]
CUIM, ZHANG WJ, ZHANGQ, XU ZQ, ZHU ZG, DUAN FP, WUR . Induced over-expression of the transcription factor OsDREB2A improves drought tolerance in rice Plant Physiology and Biochemistry, 2011,49(12):1384-1391. [本文引用: 1]
MOONSA, BAUWG, PRINSENE, VAN MM, VAN D SD . Molecular and physiology responses to abscisic acid and salts in roots of salt-sensitive and salt-tolerant indica rice varieties. Plant Physiology, 1995,107(1):177-186. [本文引用: 1]
DUAN JL, CAI WM . OsLEA3-2, an abiotic stress induced gene of rice plays a key role in salt and drought tolerance PLoS ONE, 2012,7(9):e45117. [本文引用: 1]
HU TZ . OsLEA3, a late embryogenesis abundant protein gene from rice, confers tolerance to water deficit and salt stress to transgenic rice Plant Physiology, 2008,55(4):530-537. [本文引用: 1]
KADER MA, SEIDELT, GOLLDACKD, LINDBERGS . Expressions of OsHKT1, OsHKT2, and OsVHA are differentially regulated under NaCl stress in salt-sensitive and salt-tolerant rice (Oryza sativa L.) cultivars. Journal of Experimental Botany, 2006,57(15):4257-4268. [本文引用: 1]
NATSUKO IK, NAOKIY, HIROKIY, KAORUO, HIROKIU, ALEXC, KEITAROT, HIDEOM, MIHO FK, TOMOKIH . OsHKT1;5 mediates Na+ exclusion in the vasculature to protect leaf blades and reproductive tissues from salt toxicity in rice The Plant Journal, 2017,91:657-670. [本文引用: 1]
WANGR, JINGW, XIAOL, JINY, SHENL, ZHANGW . The OsHKT1;1 transporter is involved in salt tolerance and regulated by an MYB-Type transcription factor. Plant Physiology, 2015,168(3):1076-1090. [本文引用: 1]