白菜型冬油菜类甜蛋白的筛选、克隆及其在低温胁迫下的表达

类甜蛋白是一种能在低温胁迫下诱导表达,增强植物抗逆性的关键蛋白质。本研究运用双向电泳结合质谱技术,筛选低温胁迫下陇油7号叶片差异蛋白点,从中分离出与抗寒密切相关的类甜蛋白。根据已发表植物类甜蛋白基因TLP的保守序列设计引物,采用RT-PCR扩增陇油7号的DNA,获得TLP基因开放阅读框,长度为732 bp,编码243个氨基酸的蛋白质。生物信息学分析显示,与甘蓝型油菜(Brassica napus)的蛋白质氨基酸序列同源性高达99.18%,该基因在进化上高度保守,其保守序列属于植物的GH69-TLP-SF超家族,TLP相对分子质量和理论等电点分别为26.02 k D和9.15。TLP含有一个信号肽,为亲水性蛋白,亚细胞定位预测其是在内质网中合成的蛋白。二级结构预测表明陇油7号的TLP是由不规则卷曲和延伸链组成的不稳定蛋白。实时荧光定量和半定量分析显示,在适当阈值的低温胁迫下TLP基因表达量上调,表明该基因在白菜型冬油菜陇油7号适应低温胁迫过程中发挥重要作用。     

低温胁迫下白菜型冬油菜差异蛋白质组学及光合特性分析

分离鉴定白菜型冬油菜低温差异表达蛋白质, 从蛋白质组角度揭示白菜型冬油菜抗寒机理奠定基础。以强抗寒白菜型冬油菜陇油7号为材料, 采用双向电泳和质谱分析技术, 比较低温(4°C、–4°C)和常温(25°C/20°C)下叶片蛋白质组差异;对差异蛋白进行KO和KEGG功能分析。结果表明,低温下陇油7号生长点下陷、植株匍匐, 气孔处于关闭或半关闭状态。2-DE和PDQuest8.0.1软件分析表明, 常温和4°C低温下叶片蛋白质斑点数分别726、738;相对于常温处理, 4°C低温下陇油7号叶片10个蛋白点特异表达、5个蛋白点未表达;MALDI-TOF-TOF MS质谱分析鉴定出11个蛋白质, 参与碳水化合物代谢、糖代谢、氨基酸代谢、有机酸代谢、核酸代谢、信号转导与细胞通讯等细胞过程。冰晶形态显微观察结果表明低温处理后陇油7号叶片蛋白质提取液中含有高活性抗冻蛋白(antifreeze proteins, AFPs)。鉴定的11个蛋白质中, 有5个蛋白质点与光合作用有关, 低温下陇油7号叶片1,5-二磷酸核酮糖羧化酶(RuBPCase)活性和净光合速率Pn下降。叶片Pn下降与RuBPCase表达抑制和活性降低有关, 非气孔限制是Pn下降的主要因素;高活性抗冻蛋白在白菜型冬油菜抗寒中发挥重要作用。     

白菜型油菜WUS基因的克隆与表达分析

WUS基因在植物生长点(shoot apical meristem, SAM)调控方面具有重要作用,本研究选择抗寒性强弱不同且生长点高低不同的白菜型冬油菜(Brassica rapa L.)为材料,采用RT-PCR技术克隆WUS基因并进行了表达分析,并对其功能结构域、系统进化和表达特性进行分析。结果表明,在‘陇油7号’中,WUS蛋白编码310个氨基酸,预测分子质量为23.077 kD,等电点5.58,是一个主要由无规则卷曲与延伸链组成的亲水性蛋白。荧光定量PCR分析表明,相同温度处理下,WUS基因在抗寒性较强、生长点凹陷的白菜型冬油菜中相对表达量高于抗寒性较弱、生长点凸起的白菜型冬油菜,低温(0℃, 4℃和-4℃)处理72 h后,白菜型冬油菜生长点中WUS基因在0℃均上调表达,当温度为0℃时表达量达最大,‘陇油7号’和‘天油2号’较常温上调最为显著,分别上调315.24%、287.56%。与0℃相比较,当温度降为-4℃时,各品种基因的相对表达量显著下调表达,表明WUS基因可能参与抗寒应答及白菜型冬油菜生长点对低温胁迫的响应。     

白菜型冬油菜抗寒性与生理生化特性关系

为了了解渗透物质与油菜抗寒能力的关系,本研究以2个白菜型冬油菜陇油6号和天油4号为试验材料,通过冷冻处理,对油菜根部相对电导率和叶片超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、可溶性蛋白、丙二醛(MDA)等一系列生理生化指标进行了检测。结果表明,陇油6号(DQW-1)的抗寒能力强于天油4号,陇油6号和天油4号的SOD和CAT活性、可溶性蛋白含量、MDA含量随着温度的降低均呈现出先上升后下降的变化规律。低温下耐寒性强的陇油6号能保持较高的SOD酶活性、CAT酶活性、可溶性蛋白和较低的MDA含量。因此,SOD和CAT活性、可溶性蛋白和MDA含量在一定程度上能反映白菜型冬油菜的抗寒性。     

白菜型冬油菜根系形态与抗寒性的相关分析

以10个抗寒性不同的白菜型冬油菜品种/系为试材,采用根系扫描等方法,分析不同低温胁迫(4℃,-4℃和-8℃)对白菜型冬油菜根系形态的影响,并探讨这些根系形态参数与其抗寒性(越冬率)间的关系.结果表明,随着温度降低,白菜型冬油菜品种(系)的根系表面积(root surface area,RSA)、根投影面积(root p...     

发菜组氨酸激酶基因NfHK2的克隆及其在干旱胁迫下的差异表达

由HK基因编码的组氨酸激酶是双组分信号系统的重要组成部分。为了研究发菜NfHK2基因的分子信息及其对干旱胁迫的响应,对NfHK2基因进行了克隆、序列分析以及原核表达,并分析了该基因在干旱胁迫下的差异表达。结果表明,NfHK2基因全长1 467 bp,NfHK2氨基酸序列中疏水性最强的是位于139的Leu,亲水性最强的是229位的Lys,共有49个磷酸化位点,由α-螺旋、β-折叠和无规则卷曲构成不对称的三维结构。发菜NfHK2蛋白定位于细胞质中,与筒孢藻(Cylindrospermum sp. NIES-4074)(BAZ29775.1)HK亲缘关系较近。NfHK2包含一个保守功能域His KA。将NfHK2基因转入大肠杆菌中,获得了54.2 kD的外源组氨酸激酶。随着发菜干旱胁迫程度加重,NfHK2基因的表达量逐渐升高。结果为深入挖掘发菜NfHK2的功能以及其对于干旱胁迫的调控机理提供了参考。     

冷胁迫下甘蓝型冬油菜表达蛋白及BnGSTs基因家族的鉴定与分析

谷胱甘肽S-转移酶(glutathione S-transferases, GST)参与调节植物生长、发育和逆境胁迫反应的许多方面。本研究利用双向电泳和质谱技术分析‘16VHNTS309’在冷胁迫下差异表达的蛋白质,基于GO和KEGG分析鉴定出BE、APX、SOD、GST等参与冷胁迫反应的蛋白质。利用q RT-PCR和生理指标鉴定到响应冷胁迫的关键蛋白质GST,采用同源克隆法克隆到‘16VHNTS309’的GST基因。该基因CDS长度为642bp,编码213个氨基酸,是一个不稳定蛋白,属于GST＿N＿3谷胱甘肽S-转移酶家族,与甘蓝型油菜‘ZS11’序列相似性为99.22%,与‘ZS11’和‘Vision’两者的氨基酸序列相比较发现第127位亮氨酸(L)突变为脯氨酸(P)。基因家族分析表明,在甘蓝型油菜中共鉴定到153个Bn GSTs成员,按其功能主要分为Zeta、Phi、Theta、CHQ、DHAR、Lambda和Tau这七大类型,大部分的Bn GSTs属于Phi和Tau这2种类型。系统进化将Bn GSTs分为12个亚家族,亚家族Ⅰ和Ⅷ包含了较多的成员,Bn GSTs不均匀的分布在1...     

白菜型双低冬油菜冬油7号的选育

冬油7号是以0726(20017×宁油1号)为母本、MAJA为父本杂交,经过多代选育而成的冬油菜新品种。2018年6月经中华人民共和国农业农村部登记。该品种丰产、稳产性好,品质优,抗病性强,适应性广,适宜在甘肃省中、东部及周边冬油菜主产区推广种植。     

黑龙港流域不同白菜型冬油菜品种的农艺性状评价分析

以16个白菜型冬油菜品种为试验材料,在黑龙港流域特殊的干旱气候条件下,对白菜型冬油菜品种间农艺性状的变化进行了方差分析、相关性分析和聚类分析,筛选出适宜河北省低平原地区种植的冬油菜品种,为地下水超采区域内旱作雨养和轮作休耕制度下农业种植结构调整及冬油菜品种的种植选择提供理论依据。结果表明:在黑龙港流域,选择株高较高(146.1~152.7 cm)、一次分枝数(14.5~14.9)和主花序角果数(60.7~63.1)较多、二次分枝数较少且越冬率为95%~100%的油菜品种进行种植,可以获得较高的产量。参试品种中综合性状较好的品种为‘陇油16’、‘天油1358’、‘冬油-2’,适宜在本地区及气候条件相似地区种植。     

干旱胁迫下油菜消减文库的构建及分析

以抗旱能力强的油菜Holiday为材料,以干旱处理的样品为Tester,正常水分管理(对照)为Driver构建干旱诱导的甘蓝型油菜叶片正向抑制性消减杂交 (SSH)文库。随机挑选24个阳性克隆进行PCR验证,结果表明,其中23个含有插入片段,平均大小在750 bp左右。将96个阳性克隆测序,并拼接和去除冗余,获得重叠群4条,单拷贝序列82条,平均长度为542 bp。经BlastX程序比对蛋白数据库发现,11条EST没有找到同源性序列,75条有同源序列。KOBAS分析发现,28条EST被定位到67条代谢途径中,根据P值推测,光合中的碳固定、有氧呼吸的电子供体、氮代谢、乙醛酸和二羧酸代谢在植物干旱胁迫中发挥着极为重要的作用。这些EST涉及到的功能中所占比例最大的分别是细胞器(58.82%)、结合(30.77%)和新陈代谢过程(43.72%),表明甘蓝型油菜面对干旱胁迫时,细胞器组件上的功能发挥了重要的作用,结合基因和蛋白被激活,加强了新陈代谢。     

Carbohydrate Dynamics in Leaves of Rapeseed (Brassica napus) Under Drought

Until now, the carbohydrate dynamics in leaves of rapeseed under drought have largely been unknown. For this reason, a growth chamber study was conducted to examine whether the accumulation of carbohydrates under drought stress contributes to osmotic adjustment in leaf tissue. Plants of the cultivar Titan were subjected to temporary drought in the vegetative and reproductive stages. A third variant of long‐term drought covered the period from leaf development to flowering. The level of sucrose decreased under moderate water deficit, but accumulated under severe long‐term drought. Concentrations of glucose, fructose and trehalose were significantly enhanced and that of raffinose decreased in all the variants of drought. There was no evidence that any of the carbohydrates analysed in this study or the activities of soluble acid and cell wall invertases contributed to a drought‐induced accumulation of osmolytes. The results of this study indicate that osmotic adjustment in response to drought in leaves of the rapeseed cultivar Titan is only limited. It is virtually impossible that carbohydrates function as osmoprotectants in leaves of this cultivar, rising above that of the frequently detected accumulation of proline in rapeseed under water deficit.     

Responses of differentially expressed proteins and endogenous hormones in winter rapeseed (Brassica rapa L.) roots under water deficit stress

Winter rapeseed (Brassica rapa L.) can well-adapt to environmental conditions such as barrenness, water deficit and low temperature in arid and semi-arid planting regions and is the preferred rapeseed type. In this study, we analysed changes of root system morphology, antioxidant enzyme activity, endogenous hormone contents and differentially expressed proteins (DEPs) under control (CK), slight water deficit (SWD, 50–55% of maximum field water capacity), moderate water deficit (MWD, 40–45% of maximum field water capacity) and high water deficit (HWD, 30–35% of maximum field water capacity) conditions. Winter rapeseed experienced taproot elongation, decreased taproot diameter and increased lateral root number, under water deficit stress. The accumulation of reactive oxygen species (ROS) can cause membrane system peroxidation, and antioxidant enzyme activity increases to remove ROS. Changes in jasmonic acid (JA), salicylic acid (SA), cytokinin (CTK), auxin (IAA) and gibberellin (GA) levels promote the absorption of water and minerals by driving changes in the root system architecture to resist water deficit stress. A proteomic analysis has shown that DEPs are involved in energy metabolism, antioxidation response, osmotic regulation, hormone signal transduction, protein metabolism and the stress response, and these proteins are located in the peroxisome, chloroplast, mitochondrion, cell wall, vacuole, cytoplasm, extracellular space and cell membrane. In this study, multiple DEPs (malate dehydrogenase cytoplasmic 1 OS, 14-3-3-like protein GF14 Psi, GA 3-beta-dioxygenase, glutathione reductase and jasmonate-inducible protein) were involved in the root system architecture, revealing the complexity of the root response to water deficit. Significant/extremely significant synergistic relationships were observed between antioxidant enzyme activity and endogenous hormone contents. In conclusion, ROS, endogenous hormones and stress-related proteins work synergistically to control the root system architecture of winter rapeseed roots in response to water deficit stress.     

126份芜菁种质资源地上部表型的多样性分析

芜菁是十字花科芸薹属蔬菜作物,具有耐低温、耐贫瘠等优良特征,能够在海拔4 000 m的高原地区种植.为了明晰芜菁种质资源地上部表型的特征特性,本研究以126份芜菁种质资源为材料,分析了其地上部性状的多样性.结果显示芜菁叶片呈现出比较丰富的多样性,其中叶片长度分布范围为20.8～63.17 cm,叶宽分布在6.7～23....     

等离子体对干旱胁迫下小麦种子萌发的生物学效应

用80~240 W射频等离子体处理小麦种子,在聚乙二醇（PEG-6000）模拟干旱胁迫条件下,研究了不同功率等离子体对小麦幼苗生长及抗旱性的影响。试验结果表明：不同功率的等离子体处理对干旱胁迫条件下小麦幼苗生长的影响效果不同,80~200 W处理对幼苗生长有不同程度的促进作用,根数、根长、株高、幼苗干重和根冠比各项指标均比对照有不同程度的提高,而240 W处理对小麦幼苗生长产生了抑制效应。其中160 W处理小麦种子在干旱胁迫下小麦幼苗根数、根长及干重分别比对照增加14.29%、16.94%和9.76%;丙二醛（MOD）含量及质膜透性比对照明显降低,防止或降低了膜质过氧化作用对膜的伤害;SOD、POD活性及脯氨酸含量均比对照高,表明保护性酶系统增强,一定程度上缓减了干旱胁迫对小麦幼苗的伤害,提高了其抗旱性。     

津田芜菁泛素结合酶BrUBC11基因的克隆及表达分析

为阐释津田芜菁Br UBC11基因的表达特性,克隆了津田芜菁UBC11基因的全长c DNA序列,命名为Br UBC11,Gen Bank登录号为KM396887。其c DNA全长685 bp,ORF区全长447 bp,编码148个氨基酸的开放阅读框;亚细胞定位结果显示,Br UBC11-GFP定位于细胞核内,表明Br UBC11蛋白可能在细胞核中发挥其功能。荧光定量PCR检测Br UBC11在芜菁不同组织中的表达结果表明,该基因在花瓣中表达量最高,花蕾中次之,具有组织特异性。而且Br UBC11在芜菁白色根皮中的表达受长波紫外线(UV-A)诱导。研究结果显示,在芜菁中,UBC11属于多功能基因,具有潜在的参与花发育和UV-A信号转导相关途径的功能。     

Comparisons of Isozyme Patterns in Resynthesized Amphihaploid Rapeseed (Brassica napus) and Their Parental Species Brassica campestris and Brassica oleracea

Resynthesized rapeseed plants produced by embryo culture and the progeny of their selfed parents from Brassica oleracea and Brassica campestris were analyzed by starch gel electrophoresis staining of enzymes GPI, LAP and SDH. In at least one unequivocal case it could be proved for each enzyme system studied that the alleles of both parents arc independently expressed in the newly synthesized individuals and that in the pattern of the dimeric enzyme GPI interlocus bands of the homoeologous loci arc visible. Comparisons with enzyme patterns in cultivated B. napus leads to the conclusion that in general active enzyme loci of both parental genomes are present. This is of great significance in estimating slide frequencies, degree of heterozygosity and other genetic parameters of B. nupus populations/cultivars.     

Recovery from Drought Stress at the Shooting Stage in Oilseed Rape (Brassica napus)

Periods of drought frequently affect the development of winter rape at the shooting stage in spring. A growth chamber study was conducted to determine the effects of 13 days of strong water deficit at the shooting stage followed by rewatering on oilseed rape (Brassica napus). The osmolality and the efficiency of photosystem II in leaves were measured by means of a vapour pressure osmometer and a chlorophyll fluorometer, respectively. The activities of sucrose cleaving enzymes in leaves were determined through invertase as well as sucrose synthase tests. A higher osmotic pressure was observed 2 days after water deprivation. Extracellular and vacuolar invertases as well as chlorophyll fluorescence were decreased with delay. Rewatering led to a gradual decrease in the osmotic pressure and a delayed increase in the enzyme activities as well as the photosynthetic efficiency. A linear regression model could predict osmolality in well‐watered plants based on the extracellular enzyme activity and growth stage. The ontogenetic and stress‐related pattern of extracellular invertase activity could indicate a role of this enzyme in balancing source‐sink relations. The onset of flowering was not influenced by drought, but the period of this stage was prolonged. Though, the ontogenetic stages of stressed and unstressed plants were the same at the time of harvest, the development of seed biomass was significantly depressed under stress.