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刘新宇, 葛海燕, 刘杨, 韩洪强, 陈火英
上海交通大学农业与生物学院, 上海, 200240
文献标识码: 1671-9964(2016)01-0057-06
文章编号: 1671-9964(2016)01-0057-06
收稿日期: 2015-01-7
网络出版日期: 2016-01-20
版权声明: 2016 上海交通大学期刊中心 版权所有
基金资助:
作者简介:
作者简介:刘新宇(1989-), 硕士生; 研究方向:蔬菜种质资源;
通讯作者: 陈火英(1962-), 女, 博士, 教授, 研究方向: 蔬菜种质资源.E-mail:chhy@sjtu.edn.cn
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摘要
DREB (dehydration responsive element binding protein)基因在植物非生物胁迫的应激反应中具有非常重要的作用, 本研究运用同源克隆的方法从‘YZ-14’紫茄品种中分离克隆得到了一个DREB基因, 因其与番茄和马铃薯中DREB3基因的相似度很高, 所以命名为SmDREB3。SmDREB3基因的cDNA全长为887 bp, 开放阅读框(ORF)长度为777 bp, 可以编码一个含有259个氨基酸的蛋白, 与同科作物马铃薯中DREB基因序列的相似性高达90%, 与番茄中DREB基因序列的相似性也能达到88%。成熟蛋白等电点为6.39, 分子量为28.665 kDa。RT-qPCR实验结果表明, SmDREB 基因在紫茄的各个组织中都会表达, 但表达水平具有组织特异性, 其在果皮中的表达量最高, 其次为叶和果肉, 而在根、花和茎中的表达量相对较低。本研究为深入研究茄子的抗逆机制以及培育抗逆茄子新品种方面提供了理论基础。
关键词：
Abstract
DREB has an important role in stress signal transduction pathways in plants.A cDNA fragment, named SmDREB3, was isolated from ‘YZ-14’ eggplant species using homology-based cloning method.The full length of cDNA of SmDREB is 887 bp, and the ORF is 777 bp, encoding 259 amino acids.It had 90% homologies with the sequence of potato and 88% homologies with the sequence of tomato. The mature protein isoelectric point was at pH 6.39 and molecular weight was 28.665 kDa.RT-qPCR analysis indicated that SmDREB3 was expressed in all organs.However, the expression level of SmDREB3 was tissue-specific.It expressed highest in the pericarp, followed by the leaf and sarcocarp, while it expressed relatively low in roots, stems and flowers.This study provides the theoretical basis for further researches on resilience mechanisms and cultivating new varieties that resist adverse environment.
Keywords：
植物的抗逆反应是一个由多个基因参与控制且存在多个信号转导途径的复杂网络, 参与调控此过程的基因大多为转录因子。DREB类转录因子属于AP2/EREBP(APETALA2/ethylene-responsive element binding proteins)转录因子家族, 这类转录因子不仅在植物对逆境的胁迫应答反应中起到重要的调节作用, 而且在调控植物的生长发育过程中也发挥重要的作用[1]。DREB蛋白能特异地与DRE(dehydration responsive element)顺式作用元件结合, 从而调控其下游的多个与逆境相关的基因的表达[2]。因此可以通过调控DREB基因的表达来提高植物抵抗各种不利环境的能力, 进而获得高品质的作物品种。
目前, DREB基因已经从拟南芥[3]、玉米[4]、烟草[1]、水稻[5]、棉花[6]、大豆[7]、番茄[8]和马铃薯[9]等植物中分离克隆出来, 但是在茄子中还没有研究报道。茄子是我国栽培最广、历史最悠久的果蔬之一, 在我国各个省份均有种植、生产和销售, 是城乡居民消费最多的蔬菜品种之一。茄子在生长过程中会受到缺水、高盐和低温等逆境胁迫的影响, 这些因素会影响茄子的正常生长, 甚至会导致其死亡, 从而严重影响茄子的品质和产量。因此, 对茄子DREB转录因子的研究, 对提高茄子的抗逆能力, 利用基因工程的手段培育新品种具有重要意义。
本研究从茄子中分离克隆了与茄子抗逆相关的DREB基因, 命名为SmDREB3。利用在线工具分析了该基因的核酸以及氨基酸序列, 预测了该基因编码的蛋白质的二级结构。同时, 利用Clustalx 8.31和MEGA5.0软件, 采用邻接法构建不同物种间DREB基因的系统进化树, 从而判断不同物种间DREB基因进化距离的远近。此外, 取茄子的不同组织, 如幼根、茎、叶片、花瓣、果皮和果肉进行实时荧光定量分析, 从而了解DREB基因的组织表达模式, 为进一步研究DREB基因的结构和功能打下基础。
本实验的茄子品种为‘YZ-14’, 其表型为无刺, 紫色茎, 紫色花, 紫色果实。先将材料播种于营养钵中, 置于光照培养箱中育苗, 待其长到四叶一心时, 移栽到大棚中种植。
1.2.1 总RNA的提取及cDNA第1链的合成
采用Trizol的方法提取不同组织的RNA, 材料为紫茄的幼根、茎、叶片、花瓣(呈90°)、茄子果皮和果肉。提取后的RNA分别用1.2%琼脂糖电泳和超微量分光光度计检测RNA的浓度与质量, 之后贮存于-80 ℃以备后用。cDNA第1链合成反应采用Takara公司反转录试剂盒完成。
1.2.2 SmDREB3基因cDNA全长的克隆
根据拟南芥中与成花相关DREB基因的序列信息, 在NCBI中进行BLAST比对后, 选取同属于茄科的作物, 如马铃薯、番茄和烟草的基因序列, 并获得其相应的编码区序列, 在其3'端与5'端保守区域设计可以扩增出cDNA全长的特异引物SmDREB-F和SmDREB-R(表1), 以反转录后得到的cDNA第1链作为模板, 采用宝生物工程(大连)有限公司(TaKaRa)的PrimeSTAR© Max DNA高保真酶进行扩增。
对PCR产物用1.2%琼脂糖凝胶电泳进行检测, 得到预期大小的片段后, 利用PCR产物纯化试剂盒进行纯化。随后对PCR产物加A尾, 此过程采用宝生物公司的DNA A-tailing Kit试剂盒, 再经TA克隆连接到pMD ©19-T Vector载体上, 之后用热击法把重组质粒转化到DH-5α大肠杆菌感受态中, 产物涂布于含有氨苄抗生素的平板上, 12 h后挑取单克隆摇菌, 经将菌液PCR以及1.2%琼脂糖凝胶电泳检测后, 与预期片段大小相同的阳性克隆菌液样品送至生工公司进行多次测序。
表1 本实验所用引物名称及序列
Tab.1 Primers and sequences used in this experiment
| 引物名称 Prime name | 引物序列(5’-3’) Prime sequence(5’-3’) |
|---|---|
| SmDREB3-F | ATGAATTCCCAAATCTTTTCA |
| SmDREB3-R | TTATAGAGAGGCCCAATCAAT |
| SmAction-F | TTCCTTGTATGCTAGTGGTCGTACAA |
| SmAction-R | CTCAGCACCAATGGTAATAACTTGTCC |
| SmDREB3-RT-F | GAGCAAGGTGACAGAAGTTGG |
| SmDREB3-RT-R | TCCGCCATGTCCCAAGTAGA |
1.2.3 SmDREB3基因及其编码蛋白的生物信息学分析
利用在线软件对测序得到的SmDREB3基因的序列进行分析及其蛋白质结构的预测。用ProtParam分析蛋白的分子量和等电点; 利用在线分析工具SOMPA分析SmDREB3蛋白的二级结构; 用在线软件ProtComp 9.0对亚细胞定位进行预测。利用ClustalW2进行不同物种间基因序列的比对, 并且利用MEGA5软件、采用Neighbour-joining(邻接法)构建不同物种之间DREB3基因的进化树。
1.2.4 SmDREB3基因表达模式分析
分别选取紫茄的不同组织的RNA, 分别检测RNA浓度, 每个样品取500 ng反转录成cDNA, 稀释至使引物工作的最佳倍数。以茄子SmAction-F/SmAction-R作为内参引物[10], 同时设计特异引物SmDREB3-RT-F/ SmDREB3-RT-R。荧光定量PCR反应过程采用宝生物公司的SYBR© Premix Ex Tat (Tile RnaseH Plus)试剂盒, 整个加样过程在低温的避免日光或灯光直射的黑暗环境下进行, 本实验进行3次生物重复, 即每个组织都取3个样品, 分别提取RNA, 得到的相同组织的3个RNA样品, 再用qPCR测3个RNA样品的Ct值。
以茄子cDNA为模板, 利用设计的特异引物SmDREB3-F/SmDREB3-R进行聚合酶链式反应, 得到了与预期片段大小相同的条带(图1)。将PCR产物进行胶回收、连接到pMD19-T载体上、转化到大肠杆菌感受态中、送生物公司测序, 最终得到SmDREB3的完整cDNA序列。利用NCBI网站的blastn将得到的cDNA序列进行比对, 结果表明, SmDREB3与同属于茄子的作物马铃薯和番茄DREB基因序列同源性都很高, 分别为高达90%和88%。
利用NCBI的ORF Finder在线工具, 在测序所得SmDREB3基因的cDNA序列中找到开放阅读框, 其长度为777 bp, 可以编码氨基酸259个(图2), 该蛋白的分子量(Mw)为28.665 kDa, 成熟蛋白的等电点为6.39。SmDREB3蛋白不具有跨膜结构, 不含有信号肽序列, 从而表明SmDREB3蛋白不是分泌蛋白[11](表2)。不同物种间DREB3基因的进化树图谱显示, 茄子中的SmDREB3基因与马铃薯、番茄的DREB3基因进化距离最近, 处于同一分支, 而与不同科的葡萄、小麦以及三角叶杨等进化距离较远(图3)。
图2 SmDREB3基因全长cDNA顺序及相应的氨基酸顺序ATG:起始密码子; TAA; 终止密码子
Fig.2 The full length cDNA sequence and amino acid sequence of SmDREB3ATG:indicates the initial codon:TAA indicates the termination codn
利用SOPMA在线软件预测SmDREB3蛋白的二级结构, 结果显示, 该蛋白的二级结构中含有α螺旋、ß转角、不规则卷曲和延伸链结构。其中, 不规则卷曲所占比例最大, 为45.74%; 其次是α螺旋和延伸链结构, 分别为32.95%和13.57%; ß转角最少, 仅为7.75%(图4)。利用NCBI在线分析预测SmDREB3蛋白的结构域显示, 在其靠近N端有一个DNA结合位点, 同时在83-141位是一个由59个氨基酸残基组成的典型的AP2结构域(图5)。通过ProtComp Version 9.0软件预测蛋白的亚细胞定位, SmDREB3蛋白定位在细胞核中(表3)。
图1 SmDERB3基因的克隆(a)及PCR鉴定大肠杆菌DH5α转化子(b)SmDERB3:SmDERB3基因cDNA全长; 1~4:SmDERB3基因全长DH5α转化子的PCR产物; M:DL 2 000 Marker
Fig.1 Cloning of SmDERB3(a)and PCR analysis of transformed DH5α(b)SmDERB3:Full length of SmDERB3 cDNA; 1-4:PCR products of transformed DH5α of SmDERB3cDNA; M:DL 2 000 Marker
表2 TMHMM软件跨膜分析结果
Tab.2 Outcome of transmembrane analyzing by TMHMM software
| 参数 Parameter | TMHs数量 Number of TMHs | TMHs中AAs 数目Number of AAs in TMHs | 60AAs期望值 Exp number first 60 AAs | 细胞质侧 预测值 N-in |
|---|---|---|---|---|
| 数值 | 0 | 0.00157 | 0.00134 | 0.01928 |
表3 SmDREB3蛋白亚细胞定位结果
Tab.3 Sub-cellular location of SmDREB3 protein
| Location weights 定位权重 | LocDB | PotLocDB | Neural Nets | Pentamers | Integral |
|---|---|---|---|---|---|
| 细胞核 Nuclear | 0.0 | 5.0 | 0.00 | 0.80 | 7.93 |
| 质膜 Plasma embrane | 0.0 | 0.0 | 0.96 | 0.00 | 1.44 |
| 胞外 Extracellular | 0.0 | 0.0 | 0.96 | 0.75 | 0.02 |
| 胞质 Cytoplasmic | 0.0 | 0.0 | 0.00 | 2.54 | 0.02 |
| 线粒体 Mitochondrial | 0.0 | 0.0 | 0.00 | 0.88 | 0.00 |
| 内质网 Endoplasm.retic. | 0.0 | 0.0 | 0.00 | 0.40 | 0.00 |
| 过氧化物酶体 Peroxisomal | 0.0 | 0.0 | 0.96 | 0.00 | 0.17 |
| 高尔基体 Golgi | 0.0 | 0.0 | 0.11 | 0.21 | 0.00 |
| 叶绿体 Chloroplast | 0.0 | 0.0 | 0.00 | 0.00 | 0.00 |
| 液泡 Vacuolar | 0.0 | 0.0 | 0.00 | 0.01 | 0.42 |
采用
图3 不同物种的SmDREB3基因进化树Solanum melongena:茄子; Solanum lycopersicum:番茄; Solanum tuberosum:马铃薯; Glycine max:大豆; Sophora moorcroftiana:砂生槐; Leymus chinensis:羊草; Triticum aestivum:小麦; Vitis amurensis:葡萄; Morus notabilis:川桑; Populus trichocarpa:三角叶杨
Fig.3 Phylogenetic trees of SmDREB3 nucleotides sequencefrom various species
图4 SmDREB3蛋白的二级结构注: h, α螺旋; t, β转角; c, 无规则卷曲; e, 延伸带
Fig.4 The secondary structure of SmDREB3Note: h, Alpha helix; t, Beta turn; c, Random coil; e, Extended strand
图6 SmDREB3在茄子植株不同组织的相对表达量注: 不同字母表示差异的显著性(a、b、c)(P≤0.05)
Fig.6 The relative expression of SmDREB3 in different tissues ofS.melongena LNote: Different letters show significant difference(a, b, c)(P≤0.05)
本实验采用了同源克隆的方法, 通过设计可扩增基因全长的引物, 首次从茄子并中得到了DREB基因序列。对其序列进行了初步的分析, SmDREB3基因的cDNA全长为887 bp, 开放阅读框为777 bp, 编码259个氨基酸。NCBI序列比对结果显示, SmDREB3与茄科其他作物的DREB有较高的相似性, 与马铃薯、番茄的的同源性分别为90%和88%。生物信息学的结果表明, SmDREB蛋白二级结构以不规则卷曲和α螺旋为主, 同时含有少量的延伸链和β螺旋结构。亚细胞定位预测将DREB蛋白定位于细胞核内, 这与转录因子的作用特征相符合。
DREB类转录因子在3’端和5’端的保守性较低, 而在中间有一个非常保守的AP2结构域, 其大约由59个氨基酸残基组成[11, 12]。对SmDREB蛋白结构域的分析表明, 在其83-141位的确有一个由59个氨基酸残基组成的典型的AP2结构域, 而且在靠近N端有一个DNA结合位点可以与DNA特异结合, 这证明了该基因确实属于DREB类转录因子家族。
RT-qPCR实验结果表明, SmDREB3基因在茄子的各个组织中都有表达, 但是在不同组织中的表达量差异很大。在果皮中的表达量最高, 其次是叶和果肉, 而在根、茎和花瓣中的表达量很低, 具有基因的组织表达特异性。这只是该基因在未处理茄子材料中的表达量情况, 而对其在低温、干旱以及盐胁迫下的表达量的变化情况还有待进一步研究。本实验对茄子SmDREB3基因全长的克隆, 为今后对其进行功能验证及结合分子育种技术获得抗旱、耐盐碱、耐低温的优良茄子品种提供了理论基础。
The authors have declared that no competing interests exist.
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【目的】克隆银中杨抗逆转录因 子DREB基因,为其功能的深入研究奠定基础。【方法】根据DREBAP2/EREBP保守区设计1对简并引物,采用PCR方法对银中杨(Populus albaxp)转录因子DREB基因中间片段进行克隆;再利用反向PCR方法对该基因的旁侧序列进行克隆,最终将两侧序列与中间片段拼接得到基因全长;同 时,根据基因序列设计1对特异引物,利用荧光定量PCR分析银中杨DREB基因在不同逆境胁迫中的表达情况。【结果】成功地从银中杨中克隆到1个DREB 基因,命名为PaDREB(注册号:EF582843)。该基因序列长935bp,ORF为819bp,编码272个氨基酸;同源性比较与系统发育分析证 实,银中杨DREB属于DREB转录因子家族,并与月季DREB2C具有较高的同源性;荧光定量PCR检测结果显示,盐、低温、干旱及ABA均能诱导 DREB基因的表达。【结论】首次从银中杨中分离并克隆了DREB基因,并证实其参与了银中杨对盐、低温、干旱及ABA的应答。
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Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit [J].DOI:10.1136/bjo.85.10.1248 URL [本文引用: 1] 摘要
PubMed comprises more than 21 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.
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利用玉米转录因子 ZmDREB3基因(Gene ID:EU964828.1)构建了Ubiquitin启动子驱动的植物表达载体PGM0229-ZmDREB3-EP-SPS,以EPSPS基因为抗性 筛选标记,通过花粉管通道法将农杆菌EHA105介导的植物表达载体转化到玉米自交系吉444、Mo17中,通过喷洒350mg/L草甘膦除草剂筛选,得 到7株草甘膦抗性植株,用PCR检测得到2个同时整合EPSPS标记基因和ZmDREB3目的基因的转基因株系,用PCR-Southern进一步验证, 结果呈阳性。以上结果证明外源基因已经被整合到玉米基因组中。
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Comprehensive analysis of rice DREB2-type genes that encode transcription factors involved in the expression of abiotic stress-responsive genes [J].DOI:10.1007/s00438-009-0506-y URL PMID: 20049613 [本文引用: 1] 摘要
Abstract DREB2s (dehydration-responsive element-binding protein 2s) are transcription factors that interact with a cis-acting DRE (dehydration-responsive element)/CRT (C-repeat) sequence and activate the expression of downstream genes involved in water- and heat-shock stress responses and tolerance in Arabidopsis thaliana. In this study, we performed a comprehensive analysis of all five DREB2-type genes in rice (OsDREB2 s: OsDREB2A, OsDREB2B, OsDREB2C, OsDREB2E and OsABI4) to determine which of them contribute to plant stress responses. We analysed the expression patterns of these genes under abiotic stress conditions, and we examined the subcellular localisation and transcriptional activation activity of their translational products in protoplasts. Only OsDREB2A and OsDREB2B showed abiotic stress-inducible gene expression. In addition, OsDREB2B showed nuclear specific localisation and the highest transactivation activity. OsDREB2B has functional and non-functional forms of its transcript similar to its orthologues in the grass family, and the functional form of its transcript was markedly increased during stress conditions. We analysed the splicing mechanism of OsDREB2B with transgenic rice that express the non-functional transcript and we found that the non-functional form is not a precursor of the functional form; thus, stress-inducible alternative splicing of pre-mRNA is an important mechanism for the regulation of OsDREB2B. Transgenic Arabidopsis plants overexpressing OsDREB2B showed enhanced expression of DREB2A target genes and improved drought and heat-shock stress tolerance. These results suggest that OsDREB2B is a key gene that encodes a stress-inducible DREB2-type transcription factor that functions in stress-responsive gene expression in rice.
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棉花中一个新的类DREB转录因子(GhDREB2B)的克隆, 序列特征及表达分析 [J].
<FONT face=Verdana>棉花是盐碱地改良的重要作物。文章利用盐胁迫处理棉花耐盐品种‘中棉所49’,筛选棉花EST数据库并对目标EST序列进行整合与分析,利用RT-PCR的方法,克隆了一个新的棉花类DREB转录因子,命名为<EM>GhDREB2B</EM>(GenBank登录号为GQ848094)。该基因编码351个氨基酸残基,分子量约39 kD,推导的氨基酸序列与杨树、番茄、大豆、拟南芥等物种中的DREB基因家族成员之间存在一定程度的同源性,均含AP2/ERF保守结构域,推测该基因在棉花中是一个新的DREB类转录因子。利用荧光定量RT-PCR对该转录因子表达特征的分析表明：<EM>GhDREB2B</EM>在棉花苗期经盐胁迫诱导后表达量迅速升高,在0.7%的NaCl胁迫诱导下,表达量达到最高值,但该转录因子并不受ABA的诱导。推测<EM>GhDREB2B</EM>在棉花受到干旱和盐胁迫条件下发挥重要功能。<BR></FONT>
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| [7] |
Cold-induced modulation and functional analyses of the DRE-binding transcription factor gene, GmDREB3, in soybean(Glycine max L.) [J].DOI:10.1093/jxb/ern269 URL PMID: 3071762 [本文引用: 1] 摘要
DREB (dehydration-responsive element-binding protein) transcription factors have important roles in the stress-related regulation network in plants. A DREB orthologue, GmDREB3 , belonging to the A-5 subgroup of the DREB subfamily, was isolated from soybean using the RACE (rapid amplification of cDNA ends) method. Northern blot analysis showed that expression of GmDREB3 in soybean seedlings was induced following cold stress treatment for 0.5 h and was not detected after 3 h. However, it was not induced by drought and high salt stresses or by abscisic acid (ABA) treatment. This response was similar to those of members in the A-1 subgroup and different from those of other members in the A-5 subgroup, suggesting that the GmDREB3 gene was involved in an ABA-independent cold stress-responsive signal pathway. Furthermore, analysis of the GmDREB3 promoter elucidated its cold-induced modulation. A promoter fragment containing bases 611058 to 61664 was involved in response to cold stress, and its effect was detected for 1 h after treatment, but a transcriptional repressor appeared to impair this response by binding to a cis -element in the region 611403 to 611058 at 24 h after the beginning of cold stress. Moreover, the GmDREB3 protein could specifically bind to the DRE element in vitro , and activated expression of downstream reporter genes in yeast cells. In addition, overexpression of GmDREB3 enhanced tolerance to cold, drought, and high salt stresses in transgenic Arabidopsis . Physiological analyses indicated that the fresh weight and osmolality of GmDREB3 transgenic Arabidopsis under cold stress were higher than those of wild-type controls. GmDREB3 transgenic tobacco accumulated higher levels of free proline under drought stress and retained higher leaf chlorophyll levels under high salt stress than wild-type tobacco. In addition, constitutive expression of GmDREB3 in transgenic Arabidopsis caused growth retardation, whereas its expression under control of the stress-inducible Rd29A promoter minimized negative effects on plant growth under normal growth conditions, indicating that a combination of the Rd29A promoter and GmDREB3 might be useful for improving tolerance to environmental stresses in crop plants.
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| [8] |
Expression of dehydration responsive element-binding protein-3(DREB3)under different abiotic stresses in tomato [J].DOI:10.5483/BMBRep.2009.42.9.611 URL PMID: 19788864 [本文引用: 1] 摘要
ABSTRACT We investigated the expression pattern of dehydration responsive element-binding protein-3 in tomato under different abiotic stresses. Full length LeDREB3 cDNA was isolated from tomato plant, followed by phylogenetic analysis based on deduced amino acid sequences that revealed significant sequence similarity to DREB proteins belonging to diverse families of plant species. Southern blot analysis showed duplicate copies of LeDREB3 in the tomato genome while organ-specific expression profiling indicated constitutive expression of LeDREB3 in all tested organs, which was particularly strong in flower. LeDREB3 expression was significantly induced by Nacl, drought, low temperature and H(2)O(2). Moreover, LeDREB3 was slightly regulated by treatment with ABA and MV. These observations suggest that the LeDREB3 gene may be involved in the response of the tomato plant to stress. [BMB reports 2009; 42(9): 611-616].
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| [9] |
Arabidopsis rd29A::DREB1A enhances freezing tolerance in transgenic potato [J].DOI:10.1007/s00299-007-0360-5 Magsci [本文引用: 1] 摘要
<a name="Abs1"></a>The freezing tolerance of 38 independent transgenic potato lines derived from the cultivar Desiree was tested in vitro using plantlets. The lines were transgenic for the <i>DREB1A</i> gene under control of the <i>rd29A</i> promoter, both of which were derived from <i>Arabidopsis thaliana</i>. The level of damage caused by freezing varied significantly among the transgenic clones and a non-transgenic control (cv. Desiree). Phenotypic evaluation indicated that the variable responses to freezing were attributable to genotypic variation, but freezing tolerance was not dependent on the number of insertions. Northern blot analysis using a <i>DREB1A</i> cDNA probe revealed high levels of <i>DREB1A</i> expression among the transgenic clones during the initial cold exposure at 4°C (after 2 h) and in the early stages of freezing (−20°C, 1–10 min). Furthermore, a linear correlation was detected between the level of expression and the phenotypic response for all lines except D138. Thus, in the case of potato, a significant increase in freezing tolerance was observed in vitro on a small scale following the introduction of <i>rd29A</i>::<i>DREB1A</i>. Additional testing will show whether this strategy can be used for tolerance breeding in potato and to increase the freezing tolerance of other agriculturally important crops.
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| [10] |
茄子花青素合成相关基因SmMYB的克隆与表达分析 [J].
以茄子（<em>Solanum melongena</em> L.）‘YZ14’（紫茄）和‘YZ3’（白茄）为试验材料，采用</br>同源克隆与RACE（rapid amplification of cDNA ends）相结合的方法克隆了茄子MYB 基因cDNA 及gDNA</br>全长序列，命名为<em>SmMYB</em>。序列分析结果表明：该基因cDNA 全长1 035 bp,开放阅读框837 bp，编码</br>278 个氨基酸，与辣椒（<em>Capsicum annuum</em> L.）MYB 转录因子氨基酸序列相似性达71%；成熟蛋白的等电</br>点为8.47，具有2 个典型的DNA-binding 结构域且亚细胞定位于细胞核；gDNA 全长3 834 bp,包含3 个</br>外显子及2 个内含子。荧光半定量检测结果表明：<em>SmMYB</em> 在茄子根、茎、叶、花瓣、果皮中均有表达，</br>但表达水平具有组织特异性；遮光处理后紫色茄子果皮中该基因表达量变化与花青素合成量变化趋势相</br>似。推测<em>SmMYB</em> 为一个MYB 转录因子基因，正向调控茄子花青素的合成。
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| [11] |
DOI:10.3969/j.issn.1000-6400.2014.03.019 URL [本文引用: 2] 摘要
干旱、高盐、低温等非生物逆境 胁迫严重影响植物的生长发育和作物产量。转录因子在调节植物生长发育以及对外界环境胁迫的响应方面起着重要作用。DREB类转录因子即干旱应答元件结合蛋 白是AP2/EREBP转录因子家族的一个亚家族,拥有保守的AP2结构域,能够与DRE/CRT顺式作用元件特异结合,在非生物逆境胁迫条件下调节一系 列下游胁迫诱导逆境应答基因的表达,从而提高植物耐逆性。就DREB转录因子的结构特点、表达调控以及提高转基因植株胁迫耐受性的最新研究成果进行了评 述。
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| [12] |
海岛棉DREB基因的克隆及植物表达载体的构建 [J].DOI:10.3969/j.issn.1007-8614.2010.04.007 URL [本文引用: 1] 摘要
根据GenBank上公布的棉花GhDREB1基因序列设计特异性引物,从海岛棉中克隆了一个与DREB类基因同源的cDNA序列,命名为XHDREB。序列分析表明,该cDNA序列长度为651 bp,推测含有216个氨基酸,分子量为24.6 kDa,等电点为9.46。同源性分析表明,它与GhDREB1的氨基酸序列同源性为85.19%。所编码蛋白具备DREB1转录因子的特征：含有58个氨基酸组成的AP2 DNA结合域以及两个DREB1/CBF特征氨基酸序列PKKRAGRKKFRETR和DSAWR。然后将XHDREB基因连接到pCAMBIA2300-35SOCS质粒上,构建了植物表达载体。
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