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【目的】干旱是严重影响玉米生长发育进程的一个重要因素。挖掘玉米抗旱相关基因,通过转基因功能验证和转录组分析,解析关键基因在响应干旱胁迫过程中的分子调控机制,为抗旱分子育种和遗传改良提供理论依据。【方法】以玉米自交系B104（WT）为背景材料,利用农杆菌介导方法构建过表达ZmIBH1-1转基因株系（ZmIBH1-1-OE）;通过对转基因植株进行草铵膦抗性筛选、标记基因和目的基因PCR检测,以及运用实时荧光定量PCR检测目的基因的表达情况,鉴定阳性植株和株系;以WT和ZmIBH1-1-OE转基因株系为材料,通过干旱处理（20% PEG6000）,进行表型鉴定和耐旱生理生化指标测定,验证ZmIBH1-1的抗旱功能;通过对干旱胁迫下玉米4叶期转录组的比较分析,鉴定出差异表达的基因（differentially expressed genes,DEGs）;结合DAP-seq（DNA affinity purification sequencing）分析,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的下游靶基因,利用基因组可视化软件IGV（integrative genomics viewer）分析ZmIBH1-1蛋白结合候选靶基因的位置,然后通过Dual-Luciferase试验验证ZmIBH1-1蛋白与靶基因的调控关系。【结果】通过玉米遗传转化获得12个转化事件;T3代中,能同时检测到标记基因Bar和目的基因ZmIBH1-1的植株有458个,实时荧光定量PCR检测结果表明,ZmIBH1-1-OE中ZmIBH1-1的表达量显著高于WT,株系3和株系8表达量最高,将其自交获得T4代转基因株系用于后续试验。在干旱胁迫条件下,ZmIBH1-1-OE株系存活率、叶片相对含水量、叶绿素含量、可溶性蛋白含量及其生理生化指标（超氧化物歧化酶、过氧化物酶、过氧化氢酶活性）均显著高于WT,说明玉米中过量表达ZmIBH1-1赋予玉米更高的耐旱性。转录组分析结果表明,WT与ZmIBH1-1-OE株系在干旱胁迫下有1 214个差异表达基因;Gene Ontology（GO）功能富集分析结果表明,差异表达基因主要涉及生物过程、细胞组分和分子功能,如在生物过程中主要涉及到光合作用、应激响应、脱水响应等;KEGG富集分析表明,差异表达基因主要参与植物激素信号传导、新陈代谢等过程。结合转录组显著差异表达基因和DAP-Seq分析所得到ZmIBH1-1蛋白的靶基因,初步确定ZmIBH1-1蛋白直接调控与抗旱相关的11个候选靶基因,包括2个钙信号相关基因、3个半胱氨酸代谢相关基因、1个bHLH转录因子、1个应激响应蛋白、1个谷胱甘肽转移酶、1个氧化还原过程蛋白和2个乙烯响应因子;基因组可视化结果显示ZmIBH1-1蛋白可以结合靶基因启动子区;随后通过Dual-Luciferase试验进一步表明,ZmIBH1-1蛋白可以直接作用于11个候选靶基因,其中,ZmIBH1-1蛋白可以促进ZmCa-M、ZmSYCO、ZmbHLH54、ZmGlu-r1、ZmCLPB3和ZmP450-99A2的表达,抑制ZmAGD12、ZmCYS、ZmCYSB、ZmERF-107和ZmEIN3的表达。此外,在干旱胁迫下NAC、WRKY、MYB等转录因子在ZmIBH1-1-OE和WT株系中也存在差异表达。【结论】ZmIBH1-1的过表达可以增强玉米苗期的耐旱性;ZmIBH1-1蛋白通过直接调控乙烯信号通路中的ZmERF-107和ZmEIN3的表达提高玉米的耐旱性;ZmIBH1-1蛋白通过直接调控钙信号相关基因ZmCa-M和ZmAGD12增强玉米的耐旱性;ZmIBH1-1蛋白可能通过间接调控NAC、WRKY、MYB等转录因子响应干旱胁迫。 相似文献
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Effects of LPA on the development of sheep in vitro fertilized embryos and attempt to establish sheep embryonic stem cells 
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下载免费PDF全文 ZHANG Xue-min HUANG Xiang-hua WANG Jing XING Ying LIU Fang XIANG Jin-zhu WANG Han-ning YUE Yong-li LI Xue-ling 《农业科学学报》2023,22(4):1142-1158
Lysophosphatidic acid (LPA) is a small molecule glycerophospholipid, which regulates multiple downstream signalling pathways through G-protein-coupled receptors to achieve numerous functions on oocyte maturation and embryo development. In this study, sheep in vitro fertilized embryos were applied to investigate the effects of LPA on early embryos development and embryonic stem cell establishment. At first, the maturation medium containing estrus female sheep serum and synthetic oviduct fluid (SOF) were optimized for sheep IVF, and then the effects of LPA were investigated. From 0.1 to 10 μmol L–1, LPA had no significant effect on the cleavage rate (P>0.05), but the maturation rate and blastocyst rate increased dependently with LPA concentration (P<0.05), and the blastocyst morphology was normal. When the LPA concentration was 15 μmol L–1, the maturation rate, cleavage rate and blastocyst rate decreased significantly (P<0.05), and the blastocyst exhibited abnormal morphology and could not develop into high-quality blastocyst. Besides, the exogenous LPA increases the expression of LPAR2, LPAR4, TE-related gene CDX-2
and pluripotency-related gene OCT-4 in sheep early IVF embryos with the raise of LPA concentration from 0.1 to 10 μmol
L–1. The expression of LPAR2, LPAR4, CDX-2 and OCT-4 from the LPA-0.1 μmol L–1 to LPA-10 μmol L–1 groups in early embryos were extremely significant (P<0.05), while the expression of these genes significantly decreased in 15 μmol L–1 LPA-treated embryos compared with LPA-10 μmol L–1 group (P<0.05). The inner cell mass in 15 μmol L–1 LPA-treated embryos was also disturbed, and the blastocysts formation was abnormal. Secondly, the sheep IVF blastocysts were applied to establish embryonic stem cells. The results showed that LPA made the blastocyst inoculated cells grow towards TSC-like cells. They enhanced the fluorescence intensity and mRNA abundance of OCT-4 and CDX-2 as the concentration increased from 0 to 10 μmol L–1, while 15 μmol L–1 LPA decreased OCT-4 and CDX-2 expression in the derived cells. The expression of CDX-2 and OCT-4 in the blastocyst inoculated cells of LPA-1 μmol L–1 group and LPA-10 μmol L–1 group extremely significantly increased (P<0.05), but there was significant decrease in LPA-15 μmol L–1 group compared with LPA-10 μmol L–1 group (P<0.05). Meanwhile, the protein expression of LPAR2 and LPAR4 remarkably increased after treatment of LPA at 10 μmol L–1 concentration. This study references the IVF embryo production and embryonic stem cell research of domestic animals. 相似文献
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Establishment of an efficient regeneration and genetic transformation system for Malus prunifolia Borkh. ‘Fupingqiuzi’ 下载免费PDF全文
下载免费PDF全文 LIU Yu-song WANG Hong-ying ZHAO Yong-juan JIN Yi-bo LI Chao MA Feng-wang 《农业科学学报》2022,21(9):2615-2627
Malus prunifolia Borkh. ‘Fupingqiuzi’ has significant ecological and economic value and plays a key role in germplasm development and resistance research. However, its long juvenile phase and high heterozygosity are barriers to the identification of ‘Fupingqiuzi’ progeny with excellent traits. In-vitro regeneration techniques and Agrobacterium-mediated genetic transformation systems can efficiently produce complete plants and thus enable studies of gene function. However, optimal regeneration and genetic transformation systems for ‘Fupingqiuzi’ have not yet been developed. Here, we evaluated the factors that affect the in-vitro regeneration and transformation of ‘Fupingqiuzi’. The best results were obtained when transverse leaf sections were used as explants, and they were grown in dark culture for three weeks with their adaxial sides contacting the culture medium (MS basal salts, 30 g L−1 sucrose, 8 g L−1 agar, 5 mg L−1 6-benzylaminopurine (6-BA), 2 mg L−1 thidiazuron (TDZ), and 1 mg L−1 1-naphthlcetic acid (NAA), pH 5.8). A genetic transformation system based on this regeneration system was optimized: after inoculation with A. tumefaciens solution for 8 min, 4 days of co-culture, and 3 days of delayed culture, the cultures were screened with cefotaxime (150 mg L−1) and kanamycin (15 mg L−1). We thus established an efficient regeneration and genetic transformation system for ‘Fupingqiuzi’, enabling the rapid production of transgenic material. These findings make a significant contribution to apple biology research 相似文献
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陆地棉转录因子基因GhMYB108的克隆及其在抗旱中的作用 总被引：1，自引：0，他引：1
【目的】MYB基因家族作为植物中最大的转录因子家族之一,在抵御逆境胁迫中发挥着重要的作用。克隆陆地棉MYB转录因子基因GhMYB108,并进行表达分析,验证其在干旱胁迫响应中的作用,为进一步研究GhMYB108调控陆地棉耐旱的分子机制奠定基础。【方法】根据干旱转录组数据分析,确定GhMYB108为干旱响应基因;运用聚合酶链式反应（PCR）从陆地棉根系cDNA中扩增目的基因;对GhMYB108进行基因结构特征、预测基因序列信息以及系统进化关系等生物信息学分析;利用Plant Care网站对获得的基因启动子序列进行分析;在不同逆境胁迫条件下,对GhMYB108的表达特性进行qRT-PCR分析;通过亚细胞定位确定GhMYB108蛋白在细胞中的位置;利用酵母试验验证其转录活性;使用病毒诱导的基因沉默技术（virus induced gene silencing,VIGS）沉默GhMYB108,并用qRT-PCR检测基因沉默效率。观察沉默株系在干旱处理前后的表型变化,并统计存活率,采用试剂盒测定相关生理生化指标;通过对棉花叶片喷施ABA与氟啶酮试验来分析GhMYB108与ABA的关系。【结果】从陆地棉中克隆了GhMYB108（Gh_A10G1563）,其全长879 bp,编码292个氨基酸,其蛋白质相对分子量为33.288 kD,等电点为6.037,多重序列比对和保守结构域分析,发现GhMYB108含有2个高度保守的MYB结合结构域,属于典型的R2R3型MYB转录因子。不同物种亲缘关系分析发现,GhMYB108与AtMYB108、AtMYB78和AtMYB2的同源性较高,属于同一亚族,且已有研究发现AtMYB108、AtMYB78和AtMYB2与干旱或ABA信号通路相关。GhMYB108定位于细胞核,且具有转录激活活性。在干旱和对照植株中,GhMYB108均在根中表达量最高,茎中表达量最低,并且受自然干旱、18% PEG 6000模拟干旱、盐胁迫和低温等非生物胁迫诱导表达。GhMYB108沉默之后,在自然干旱条件下,沉默植株出现临界表型,与对照相比,其萎蔫更严重,且存活率降低,一些生理生化指标也发生显著变化,如叶片失水率加快,丙二醛含量升高,叶片相对含水量和脯氨酸含量减少,过氧化氢酶（CAT）和过氧化物酶（POD）活性降低,且通过DAB与NBT染色发现植物体积累了更多过氧化氢（H2O2）和超氧阴离子（O2-）。通过对棉花叶片喷施激素ABA或氟啶酮发现GhMYB108可受ABA信号的正调控。【结论】GhMYB108正调控棉花抗旱性,且受ABA信号的正调控。 相似文献
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Grain yield,nitrogen use efficiency and physiological performance of indica/japonica hybrid rice in response to various nitrogen rates 下载免费PDF全文
下载免费PDF全文 ZHOU Qun YUAN Rui ZHANG Wei-yang GU Jun-fei LIU Li-jun ZHANG Hao WANG Zhi-qin YANG Jian-chang 《农业科学学报》2023,22(1):63-79
Utilizing the heterosis of indica/japonica hybrid rice (IJHR) is an effective way to further increase rice grain yield. Rational application of nitrogen (N) fertilizer plays a very important role in using the heterosis of IJHR to achieve its great yield potential. However, the responses of the grain yield and N utilization of IJHR to N application rates and the underlying physiological mechanism remain elusive. The purpose of this study was to clarify these issues. Three rice cultivars currently used in rice production, an IJHR cultivar Yongyou 2640 (YY2640), a japonica cultivar Lianjing 7 (LJ-7) and an indica cultivar Yangdao 6 (YD-6), were grown in the field with six N rates (0, 100, 200, 300, 400, and 500 kg ha–1) in 2018 and 2019. The results showed that with the increase in N application rates, the grain yield of each test cultivar increased at first and then decreased, and the highest grain yield was at the N rate of 400 kg ha–1 for YY2640, with a grain yield of 13.4 t ha–1, and at 300 kg ha–1 for LJ-7 and YD-6, with grain yields of 9.4–10.6 t ha–1. The grain yield and N use efficiency (NUE) of YY2640 were higher than those of LJ-7 or YD-6 at the same N rate, especially at the higher N rates. When compared with LJ-7 or YD-6, YY2640 exhibited better physiological traits, including greater root oxidation activity and leaf photosynthetic rate, higher cytokinin content in the roots and leaves, and more remobilization of assimilates from the stem to the grain during grain filling. The results suggest that IJHR could attain both higher grain yield and higher NUE than inbred rice at either low or high N application rates. Improved shoot and root traits of the IJHR contribute to its higher grain yield and NUE, and a higher content of cytokinins in the IJHR plants plays a vital role in their responses to N application rates and also benefits other physiological processes. 相似文献
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【目的】独行菜在种子萌发和幼苗生长阶段可耐受一定程度的低温胁迫,研究独行菜的低温耐受机制。【方法】设计兼并引物,从独行菜冷诱导叶片的cDNA中克隆获得LaCBF3和LaCOR15a基因核心片段。利用半定量RT-PCR法分析两个基因在不同胁迫处理下的表达情况。【结果】获得442 bp LaCBF3及405 bp LaCOR15a核心片段,经比对与拟南芥CBF3、COR15a基因核心片段相似性分别为84.38%、81.8%。半定量RT-PCR结果显示在独行菜幼苗中,LaCOR15a基因不仅响应低温胁迫,还能迅速响应高盐、干旱及ABA处理,不同条件下表达各有差异。LaCBF3仅对低温胁迫响应迅速。【结论】LaCBF3、LaCOR15a在独行菜幼苗响应低温胁迫的分子机制中具有重要的调控作用,且LaCOR15a基因也应答干旱、高盐等非生物胁迫。 相似文献
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Overexpression of a Cytosolic Ascorbate Peroxidase Gene,OsAPX2, Increases Salt Tolerance in Transgenic Alfalfa 下载免费PDF全文
下载免费PDF全文 Alfalfa (Medicago sativa L.) is an important forage crop in the world and it is of great significance for the improvement of its salt tolerance. To improve salt tolerance in alfalfa, a rice ascorbate peroxidase gene (OsAPX2) was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation with marker gene bar. The different T-DNA insertions in T1 transgenic alfalfa were identified by Southern hybridization. Three independent T2 transgenic lines were selected for stress analysis and the results showed that all of them were salt tolerant compared with wild-type plants. The transgenic plants had low levels of H2O2, malondialdehyde and relative electrical conductivity under salt and drought stresses. Moreover, the contents of chlorophyll and proline, and APX activity were high in transgenic plants under salt and drought stresses. Taken together, the overexpression of OsAPX2 enhances salt tolerance in alfalfa through scavenging reactive oxygen species. 相似文献
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【目的】 研究盐爪爪液泡膜Na+/H+反向运输载体KfNHX1 (AY825250) 基因的耐盐功能,为耐盐育种提供候选基因。【方法】 采用农杆菌介导花序浸染的方法,将KfNHX1转入拟南芥中,结合基因组PCR和RT-PCR方法鉴定符合3∶1分离比的转基因株系;利用在盐胁迫下的萌发率、根长和表型分析,结合原子吸收分光光度计法测定叶片的Na+、K+含量,推断其耐盐性。【结果】 对抗生素筛选符合3∶1的转基因纯合株系进行基因组PCR和RT-PCR分析,证实KfNHX1基因在拟南芥基因组中整合和表达。盐胁迫下转基因株系的拟南芥种子的萌发率和根长明显高于野生型。200 mM NaCl胁迫处理15 d的拟南芥成苗,相较野生型叶片萎黄和死亡,转基因植株的生长表型较好,且积累了较高的Na+和K+。外源ABA的处理下,转基因植株的发芽率和生长表型也好于野生型。【结论】 盐爪爪(Kalidium foliatum)是一种藜科(Chenopodiaceae)盐生灌木,对盐的耐受性很强。液泡膜Na+/H+反向运输体(NHX)是在离子稳态中起重要作用的膜蛋白,通过调节胞间离子的跨膜转运来维持细胞内离子和pH平衡。盐生植物盐爪爪KfNHX1能够提高转基因拟南芥的耐盐性,具有提高植物耐盐性的潜力。 相似文献
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白桦BpCHS3转基因植株耐盐性分析 总被引：1，自引：0，他引：1
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Sugar content is a determinant of apple (Malus×domestica Borkh.) sweetness. However, the molecular mechanism underlying sucrose accumulation in apple fruit remains elusive. Herein, this study reported the role of the sucrose transporter MdSUT2.1 in the regulation of sucrose accumulation in apples. The MdSUT2.1 gene encoded a protein with 612 amino acid residues that could be localized at the plasma membrane when expressed in tobacco leaf protoplasts. MdSUT2.1 was highly expressed in fruit and was positively correlated with sucrose accumulation during apple fruit development. Moreover, complementary growth assays in a yeast mutant validated the sucrose transport activity of MdSUT2.1. MdSUT2.1 overexpression in apples and tomatoes resulted in significant increases in sucrose, fructose, and glucose contents compared to the wild type (WT). Further analysis revealed that the expression levels of sugar metabolism- and transport-related genes SUSYs, NINVs, FRKs, HXKs, and TSTs increased in apples and tomatoes with MdSUT2.1 overexpression compared to WT. Finally, unlike the tonoplast sugar transporters MdTST1 and MdTST2, the promoter of MdSUT2.1 was not induced by exogenous sugars. These findings provide valuable insights into the molecular mechanism underlying sugar accumulation in apples. 相似文献