苹果乙烯不敏感基因EIN2片段的克隆及序列分析

分别以苹果果实总DNA和cDNA为模板,采用PCR、RT-PCR方法扩增、克隆乙烯不敏感基因(ethyleneinsensitive 2,EIN2),并利用生物信息学方法分析其核苷酸序列和蛋白质结构。结果表明:(1)以DNA和cDNA为模板的扩增结果完全相同,扩增的EIN2基因片段为4 378bp,尚未发现有内含子,开放阅读框全长3 282bp,编码1 093个氨基酸;苹果EIN2相对分子质量为118.9kD,等电点为5.52,其蛋白可能为脂溶性疏水蛋白。(2)所克隆苹果EIN2基因编码的氨基酸序列与拟南芥(AAD41077.1)、碧桃(ACY78397.1)和葡萄(CAN66374.1)EIN2基因编码的氨基酸序列一致性分别为52%、79%、62%。(3)构建的EIN2基因进化树显示,拟南芥、小盐芥、甜瓜、杨毛果EIN2基因亲缘关系较近,聚为一类;葡萄为一类;蒺藜苜蓿为一类;碧桃、矮牵牛、西红柿聚为一类;苹果单独为一类。而且苹果EIN2基因与碧桃等同源基因的亲缘关系相对较近,与拟南芥、小盐芥同源基因的亲缘关系相对较远。     

家蝇变应原Tropomyosin基因的克隆、序列分析及诱导表达

对家蝇变应原原肌球蛋白(Tropomyosin)基因进行同源克隆,序列分析;构建原核表达载体并在大肠杆菌中表达。从家蝇幼虫cDNA文库中筛选获得Tropomyosin基因。以该基因的cDNA文库质粒为模板,进行PCR扩增,获得家蝇Tropomyosin完整编码序列。运用生物信息学方法对该基因及其编码蛋白的基本理化性质、信号肽、二级结构、三级结构、抗原表位和亚细胞定位等方面进行预测和分析。构建pEASY-E1-Tropomyosin重组质粒,转化到大肠杆菌OrigamiB(DE3)中进行诱导表达。Tropomyosin基因ORF全长828 bp,编码275个氨基酸,理论分子量31.6 kD;等电点为4.65,具有Tropomyosin家族的蛋白保守结构域。成功构建重组原核表达pEASY-E1-Tropomyosin并诱导表达重组蛋白。     

马铃薯硝态氮转运蛋白基因的克隆及表达分析

该研究以马铃薯双单倍体‘DM’为材料,克隆到高亲和性硝态氮转运蛋白基因StNRT2.1的全长cDNA(JGI登录号PGSC0003DMT400002924),并对其进行表达模式和生物信息学分析,为深入探索StNRT2.1基因的生物学功能以及提高马铃薯对氮素的利用效率奠定理论基础。结果表明:(1)通过同源克隆与PCR扩增获得StNRT2.1基因cDNA全长片段,并构建pCEGFP-StNRT2.1表达载体;测序结果显示其实际所编码的蛋白质序列与数据库中目的基因蛋白质序列完全一致,表明成功克隆到StNRT2.1基因且未出现错义突变。(2)StNRT2.1基因位于马铃薯第11号染色体,cDNA序列全长1 593 bp,编码530个氨基酸,预测蛋白相对分子质量约为57.60 kD,理论等电点为9.36。(3)生物信息学分析显示,StNRT2.1由20种氨基酸组成,其中甘氨酸(Gly)所占比例最多,达到10.8%,并且主要由228个α-螺旋、27个β-折叠、87个延伸链和188个无规则卷曲构成;StNRT2.1存在功能保守结构MFS_1(PF07690)和12个跨膜螺旋结构域,且N端和C端均位于细胞膜内; StNRT2.1位于质膜上且不具有信号肽,可能为非分泌型膜蛋白。(4)以氮充足(7.5 mmol/L)水平作为对照,马铃薯幼苗经无氮(0 mmol/L)和低氮(0.75 mmol/L)处理3周后呈现出叶片发黄及植株矮化等明显表型差异。(5)qRT-PCR结果显示,在无氮条件下,马铃薯根组织中StNRT2.1基因表达量升高3.98倍,说明StNRT2.1可能为诱导型高亲和转运蛋白。     

苎麻转录因子基因BnMYB3的克隆及表达分析

该研究采用RACE技术,从苎麻中克隆到1个MYB转录因子基因(BnMYB3)的全长cDNA序列(GenBank登录号为MF741320.1)。生物信息学分析表明,BnMYB3基因cDNA全长为1 216bp,包括900bp编码区序列,编码含有299个氨基酸的蛋白,其分子量约为33.63kD,理论等电点为9.16;该蛋白质含有2个典型的MYB结构域,属于R2R3-MYB。从苎麻基因组中克隆了BnMYB3基因1 681bp启动子序列,该序列包含ABRE、GARE-motif、CGTCA-motif和TGACG-motif等多个逆境相关的顺式作用元件。实时荧光定量PCR分析表明,BnMYB3为组成型表达基因,在茎和叶中的表达量显著高于根;BnMYB3基因能够响应镉胁迫,且表达量随镉胁迫处理时间和处理浓度的增加而显著上升。     

酿酒葡萄分支酸合成酶基因(VvCS)的克隆与表达分析

本研究以酿酒葡萄(Vitis vinifera)品种赤霞珠(Cabernet Sauvignon)及霞多丽(Chardonnay)为试材,采用in silico克隆和分子克隆相结合的策略,从果实中克隆到分支酸合成酶基因,命名为VvCS。该基因的cDNA编码区全长1312bp,编码436个氨基酸残基,预测其编码蛋白质分子量为46.9kD,等电点为7.8;生物信息学分析显示VvCS的DNA全长7117bp,包含13个外显子和12个内含子,定位于葡萄的第13号染色体上。VvCS编码的蛋白与其它植物来源的分支酸合成酶在氨基酸水平上的同源性为75%左右;实时荧光定量PCR分析表明VvCS在葡萄果实、茎、叶和叶柄组织中均有表达,且在果皮、果肉和种子中的表达变化趋势相似,与盛花后5周的果实相比,盛花后11周果实各部位中VvCS表达丰度均有不同程度增加。     

染色体7q32-ter鼻咽癌相关基因的初步研究

为克隆7q32-ter区域等位基因杂合性丢失最小共同缺失区的鼻咽癌相关基因.以STS D7S509为探针,PCR法筛选位于该区的细菌人工染色体(BAC)克隆,应用EST介导的定位-候选克隆策略并结合生物信息学筛选出在鼻咽癌细胞株和活检组织中表达增强的EST AA773454,cDNA克隆测序和生物信息学资源获取全长cDNA,DNA印迹和甲基化分析研究其表达增强的机制.结果表明,克隆的NAG18基因cDNA全长802 bp,编码227个氨基酸,定位于胞核.该基因分别与人、鼠TAXREB107基因及RPL6基因高度同源.其表达增强的机制不是基因拷贝数的丢失和甲基化位点的改变.可以断定NAG18是定位于7q32-ter最小共同缺失区的鼻咽癌相关基因,它是一高度保守的基因,参与了DNA的转录活化.     

家蚕细胞凋亡相关基因BmICAD的克隆、序列和功能分析及其在精巢中特异表达的初步研究

DREP-1基因在果蝇的细胞凋亡过程中对DNA的降解有重要调控作用。本研究将该基因序 列在家蚕EST数据库中进行同源性检索,把检索到的EST序列进行电子延伸和克隆重叠群拼接,根据拼接结果设计引物进行PCR扩增并克隆测序验证,首次成功克隆了家蚕第一个ICAD基因BmICAD的cDNA： 该cDNA全长844 bp,ORF长522 bp, 编码含有174个氨基酸的蛋白;预测分子量为19.6kD,等电点为4.23,所编码蛋白与果蝇DREP-1的一致性(identity)为36%;虽然Northern印记杂交未检测到信号,但是RT-PCR结果显示,该基因在精巢中特异表达。     

马铃薯StSnRK2.1基因克隆与生物信息学分析

SnRK2基因对植物的逆境胁迫具有重要的调节作用,以马铃薯‘陇薯3号’(Solanum tuberosum)为试材,采用RT-PCR方法从马铃薯试管苗中克隆得到1个SnRK2.1基因cDNA,命名为StSnRK2.1,提交GenBank注册,注册号为JX280911。通过生物信息学分析,该基因开放阅读框全长1 008 bp,编码335个氨基酸。预测蛋白质分子量约为37.77 kD,等电点为5.37,蛋白质二级结构预测α-螺旋42.39%,延伸链16.42%,β-折叠7.46%,无规卷曲33.73%,具有疏水性,为膜内蛋白。亚细胞定位显示该基因出现在细胞质及微体中的可能性较大。肽链可能有7处丝氨酸磷酸化位点,2处苏氨酸磷酸化位点,以及3处酪氨酸磷酸化位点,因此推测该基因在植物抗逆中有重要的作用。     

小鼠睾丸生精细胞凋亡相关基因SRG2的分子克隆及其在隐睾中的表达特征

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段(BE644542)入手,利用网上生物信息学克隆了SRG2基因全长,GenBank登录号为AF395083。从小鼠睾丸cDNA文库中分离出该基因完整阅读框cDNA,SRG2基因的cDNA全长为1088bp,为编码295个氨基酸、分子量为33579kD、等电点为9．64的蛋白质,与人类同源基因TSARG2相似性为78％,而与其他已知蛋白质无明显同源性。RT-PCR结果表明：该基因只在睾丸中有高表达。应用新型的分子信标检测该基因在不同时期隐睾中的mRNA表达水平,发现该基因呈明显上调,证明该基因在隐睾的发生发展中具重要作用。     

小鼠睾丸生精细胞凋亡相关基因SRG2的分子克隆

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 4 4 5 4 2 )入手 ,利用网上生物信息学克隆了该基因全长 ,GenBank登录号为AF395 0 83。从小鼠睾丸cDNA文库中分离出该基因完整阅读框cDNA ,SRG2基因的cDNA全长为 10 5 8bp ,编码由 2 95个氨基酸组成、分子量为 335 79、等电点为 9.6 4的蛋白质 ,与人类同源基因TSARG2相似性为 78% ,与已知蛋白质无明显同源性。RT PCR结果表明该基因只在睾丸中有高表达。     

Moonlighting kinases with guanylate cyclase activity can tune regulatory signal networks

Guanylate cyclase (GC) catalyzes the formation of cGMP and it is only recently that such enzymes have been characterized in plants. One family of plant GCs contains the GC catalytic center encapsulated within the intracellular kinase domain of leucine rich repeat receptor like kinases such as the phytosulfokine and brassinosteroid receptors. In vitro studies show that both the kinase and GC domain have catalytic activity indicating that these kinase-GCs are examples of moonlighting proteins with dual catalytic function. The natural ligands for both receptors increase intracellular cGMP levels in isolated mesophyll protoplast assays suggesting that the GC activity is functionally relevant. cGMP production may have an autoregulatory role on receptor kinase activity and/or contribute to downstream cell expansion responses. We postulate that the receptors are members of a novel class of receptor kinases that contain functional moonlighting GC domains essential for complex signaling roles.     

Promotive Effects of the Peptidyl Plant Growth Factor,Phytosulfokine-α, on the Growth and Chlorophyll Content of Arabidopsis Seedlings under High Night-time Temperature Conditions

In order to investigate the function of the peptidyl plant growth factor, phytosulfokine-α (PSK-α), in plants, we examined the effect of PSK-α on the growth and chlorophyll content of Arabidopsis seedlings under high night-time temperature conditions. Although exposure to high night-time temperatures markedly reduced the fresh weight and chlorophyll content of the seedlings, these parameters in the plants supplied with PSK-α remained at the same levels as those of non-treated controls. These effects were not apparent when [2-5]PSK, Tyr-SO₃H and kinetin were similarly supplied. The results suggest that PSK-α not only promotes cell proliferation, but may aid plants in their tolerance of heat stress.     

Further analogues of plant peptide hormone phytosulfokine-alpha (PSK-alpha) and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor of structure H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In studies on the structure/activity relationship of PSK-alpha the synthesis was performed of a series of a further 23 analogues modified in position 1, 3 or 4 as well as simultaneously in positions 1 and 3 of the peptide chain. Peptides were synthesized by the solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK-alpha according to the test of Matsubayashi et al. Among these peptide analogues, [H-Phe(4-Cl)1]-PSK-alpha (IV), [H-Phe(4-I)1]-PSK-alpha (VII), and [Phe(4-Cl)3]-PSK-alpha (XI) retained 30% PSK-alpha activity. Analogue [Tyr(PO3H2)3]-PSK-alpha (IX) showed 10% of PSK-alpha activity.     

植物肽类信号分子

近年来研究表明植物中也存在着肽类信号分子,类似于动物的肽激素。目前可分成三类：即EN-OD40、植物硫动蛋白（phytosulfokine）和系统素。ENOD40影响着细胞分裂,可能是参与控制了细胞对生长素的反应所致。植物硫动蛋白最初从芦笋叶肉细胞培养中分离,是一种减数分裂诱导因子,为细胞增殖必需,但具体机制还不清楚。系统素的研究较为深入,巳提出了它所激发的植物伤诱信号传导过程和模型。     

Proteolytic processing of a precursor protein for a growth-promoting peptide by a subtilisin serine protease in Arabidopsis

Phytosulfokines (PSKs) are secreted, sulfated peptide hormones derived from larger prepropeptide precursors. Proteolytic processing of one of the precursors, AtPSK4, was demonstrated by cleavage of a preproAtPSK4-myc transgene product to AtPSK4-myc. Cleavage of proAtPSK4 was induced by placing root explants in tissue culture. The processing of proAtPSK4 was dependent on AtSBT1.1, a subtilisin-like serine protease, encoded by one of 56 subtilase genes in Arabidopsis. The gene encoding AtSBT1.1 was up-regulated following the transfer of root explants to tissue culture, suggesting that activation of the proteolytic machinery that cleaves proAtPSK4 is dependent on AtSBT1.1 expression. We also demonstrated that a fluorogenic peptide representing the putative subtilase recognition site in proAtPSK4 is cleaved in vitro by affinity-purified AtSBT1.1. An alanine scan through the recognition site peptide indicated that AtSBT1.1 is fairly specific for the AtPSK4 precursor. Thus, this peptide growth factor, which promotes callus formation in culture, is proteolytically cleaved from its precursor by a specific plant subtilase encoded by a gene that is up-regulated during the process of transferring root explants to tissue culture.     

Plant peptide hormone phytosulfokine (PSK-alpha): synthesis of new analogues and their biological evaluation.

Phytosulfokine-alpha (PSK-alpha), a sulfated growth factor (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) universally found in both monocotyledons and dicotyledons, strongly promotes proliferation of plant cells in culture. In our studies on structure/activity relationship in PSK-alpha the synthesis of a series of analogues was performed: [H-D-Tyr(SO3H)1]- (9), [H-Phe(4-SO3H)1]- (10), [H-D-Phe(4-SO3H)1]- (11), [H-Phg(4-SO3H)1]- (12), [H-D-Phg(4-SO3H)1]- (13), H-Phe(4-NHSO2CH3)1]- (14), [H-D-Phe(4-NHSO2CH3)1]- (15), [H-Phe(4-NO2)1]- (16), [H-D-Phe(4-NO2)1]- (17), [H-Phg(4-NO2)1]- (18), [H-D-Phg(4-NO2)1]- (19), [H-Hph(4-NO2)1]- (20), [H-Phg(4-OSO3H)1]- (21), [Phe(4-NO2)3]- (22), [Phg(4-NO2)3]- (23), [Hph(4-NO2)3]- (24), [H-Phe(4-SO3H)1, Phe(4-SO3H)3]- (25) [H-Phe(4-NO2)1, Phe(4-NO2)3]- (26), [H-Phg(4-NO2)1, Phg(4-NO2)3]- (27), [H-Hph(4-NO2)1, Hph(4-NO2)3]- (28) and [Val3]- PSK-alpha (29). For modification of the PSK-alpha peptide chain the novel amino acids and their derivatives were synthesized, such as: H-L-Phg(4-SO3H)-OH (1), H-D-Phg(4-SO3H)-OH (2), Fmoc-Phg(4-SO3H)-OH (3), Fmoc-D-Phg(4-SO3H)-OH (4), Boc-Phg(4-NHSO2CH3)-OH (5), Boc-D-Phg(4-NHSO2CH3)-OH (6) Boc-Phe(4-NHSO2CH3)-OH (7), and Boc-D-Phe(4-NHSO2CH3)-OH (8). Peptides were synthesized by a solid phase method according to the Fmoc procedure on a Wang-resin. Free peptides were released from the resin by 95% TFA in the presence of EDT. All peptides were tested by competitive binding assay to the carrot membrane using 3H-labelled PSK according to the Matsubayashi et al. test.     

PSKR1 and PSY1R-mediated regulation of plant defense responses

Plant peptide signaling is an upcoming topic in many areas of plant research. Our recent findings show that the tyrosine sulfated peptide receptors PSKR1 and PSY1R are not only involved in growth and development but also in plant defense. They modulate salicylate- and jasmonate-dependent defense pathways in an antagonistic manner and this phenomenon might be dependent on the age and developmental stage of the plant. Here we discuss how the endogenous peptides might integrate growth, wounding, senescence and the opposing defense pathways against biotrophic and necrotrophic pathogens for increased fitness of the plant.     

Plant signalling peptides

Biochemical and genetic studies have identified peptides that play crucial roles in plant growth and development, including defence mechanisms in response to wounding by pests, the control of cell division and expansion, and pollen self-incompatibility. The first two signalling peptides to be described in plants were tomato systemin and phytosulfokine (PSK). There is also biochemical evidence that natriuretic peptide-like molecules, immunologically-related to those found in animals, may exist in plants. Another example of signalling peptide is ENOD40, a product of a gene, which became active early in the root nodulation process following Rhizobium infection of legumes. Other predicted bioactive peptides or oligopeptides have been identified by means of genetic, rather then biochemical methods. The Arabidopsis CLAVATA3 protein is required for the correct organization of the shoot apical meristem and the pollen S determinant S-locus cysteine-rich protein (SCR) also called S-locus protein 11, SP11). The plant signalling peptides discovered so far are involved in various processes and play an important role in communication between cells or organs, respectively. This review will focus on these peptides and their role in intercellular signalling.