沙冬青脱水素基因提高转基因紫花苜蓿的耐旱性

为鉴定转基因紫花苜蓿的耐旱能力,本研究以转基因紫花苜蓿T0代植株为材料,通过干旱胁迫,从转基因植株的表型、生理指标和分子水平等层面检测转基因植株的抗旱特性,试验表明,干旱胁迫后转基因苜蓿和对照的茎、叶片全部干枯,而复水后转基因苜蓿大部分恢复生长,对照几乎全部死亡。干旱胁迫后苜蓿叶片的Pro和MDA含量均随处理时间延长而逐渐增加,转基因植株叶片的Pro含量高于对照,而其MDA含量低于对照植株,且二者均达到了显著差异;转基因苜蓿的离体叶片失水率也明显低于对照。在20%PEG胁迫下,植株体内ProDH和P5CS基因的相对表达量都是先升后降,转基因植株和对照中ProDH和P5CS基因的表达量显著增加,且二者在转基因植株与对照间存在显著差异。这表明,在干旱胁迫条件下,由于AmDHN基因在紫花苜蓿中的过量表达,可能引起植株体内脯氨酸的大量积累,并进一步诱导脯氨酸合成相关基因的表达,最终引起植株抗旱性的提高,因此,推测转基因苜蓿的耐旱性比对照强。     

紫花苜蓿F-box蛋白基因MsFTL的克隆及功能分析

FTL(F-box Triple LRR protein)是F-box蛋白家族的成员,具有F-box保守结构域,在植物抵御逆境胁迫过程中起重要作用。本研究参考低温胁迫下紫花苜蓿转录组数据设计引物,通过RT-PCR克隆获得紫花苜蓿MsFTL基因,该基因的全长1422 bp,编码473个氨基酸。该蛋白含有1个F-box结构域及3个LRR重复。系统进化分析表明,MsFTL与蒺藜苜蓿XP_003626345.1 F-box/FBD/LRR-repeat protein亲缘关系最近。两者蛋白序列比对发现共有11个差异位点。在低温、盐、干旱以及外源ABA处理下,MsFTL基因受到诱导,表达量上调。构建植物过表达载体pCBM-MsFTL,通过农杆菌介导法转化烟草。对经过抗性筛选、PCR和Real-time PCR验证的转基因植株进行低温抗性鉴定。在-4℃低温胁迫下,野生型烟草叶片出现了明显的萎蔫失水现象,而转基因烟草萎蔫程度相对较轻。生理检测结果表明,4℃处理24 h之后,转基因烟草的可溶性蛋白含量、可溶性糖含量、SOD活性,CAT活性高于野生型,MDA含量低于野生型。本研究表明,MsFTL基因在提高植物对低温胁迫的抗性方面具有重要的作用。     

转天山雪莲sikPIP3基因烟草的获得及抗逆性鉴定

利用PCR技术从实验室建立的天山雪莲DNA文库中克隆了天山雪莲质膜水孔蛋白基因sikPIP3,构建了植物表达载体pBI121-sikPIP3,通过农杆菌介导法转化烟草品种NC89,经PCR和RT-PCR检测证明目的基因成功导入并得到了表达,采用水分胁迫进行抗旱分析和采用冷冻胁迫进行抗寒性分析。结果显示:(1)克隆出具有水孔蛋白基因特性的sikPIP3基因。(2)经断水干旱处理,转基因烟草的生长表型优于野生型烟草,特别是在断水9d的情况下,野生型烟草已经完全萎蔫,转基因烟草萎蔫症状较轻;生理指标测量结果显示,转基因烟草的相对电导率和MDA的含量低于野生型烟草,相对含水量和CAT活性高于野生型烟草。试验表明转sikPIP3烟草的抗旱性高于野生型烟草。(3)经不同温度胁迫处理,转基因烟草的生长表型优于野生型烟草,特别是在-4℃冷处理6h情况下,野生型烟草已经完全萎蔫,转基因烟草只出现少量伤斑;生理指标分析结果表明,转基因烟草的相对电导率和MDA的含量低于野生型烟草,CAT活性高于野生型烟草。试验表明转sikPIP3烟草的抗寒性高于野生型烟草。综合结果表明:sikPIP3基因在抗逆基因工程方面具有较高的应用前景。     

AtNDPK2 基因转化敖汉苜蓿及其耐盐性分析

植物核苷二磷酸激酶(NDPKs),属于蛋白激酶家族,参与植物的多种生理生化过程,在植物响应非生物胁迫过程中发挥重要作用。NDPK2基因转化紫花苜蓿,对提高紫花苜蓿的耐逆性具有重要意义。试验以"敖汉"苜蓿下胚轴的再生系统为基础,通过农杆菌介导法将SWPA2驱动的AtNDPK2基因转入紫花苜蓿下胚轴,获得抗性植株。通过PCR初步检测证明该基因已经整合到"敖汉"苜蓿的基因组,阳性植株转化率为23.3%。不同浓度NaCl胁迫测定转基因植株的SOD、POD活性及质膜相对电导率、MDA含量,结果发现在0.4%~1.0%NaCl胁迫下转基因植株的SOD、POD酶活性显著高于对照,相对电导率、MDA含量显著的低于对照,进一步说明AtNDPK2基因的转入增强了"敖汉"苜蓿的耐盐性。     

蒙古冰草MwMYB4基因功能鉴定

MwMYB4基因是从蒙古冰草中克隆得到的MYB类转录因子家族成员之一。该研究以转MwMYB4基因的拟南芥后代为材料,通过在干旱和低温胁迫下对转基因植株进行表型分析、理化指标测试和分子鉴定,分析并验证MwMYB4基因的功能。结果显示:(1)蒙古冰草MwMYB4基因已成功整合到转基因拟南芥T_1代的基因组中并实现转录水平的表达。(2)转基因拟南芥T_2代植株在干旱胁迫条件下,转基因植株叶片枯黄程度较轻,相对电导率较野生型变化幅度低,脯氨酸含量明显高于野生型对照,且MwMYB4基因的表达量随干旱胁迫时间延长而增加。(3)在低温胁迫条件下,转基因拟南芥叶片的枯白程度明显低于野生型,且MwMYB4基因的表达量随低温胁迫时间增加而增加。研究表明,过量表达蒙古冰草MwMYB4基因能够提高转基因拟南芥对干旱和低温的耐受性,该基因可能在干旱胁迫和低温胁迫调控机制中发挥调控作用,可作为改良农作物和其他牧草抗旱、抗寒性的重要候选基因。     

转ZjAPX基因拟南芥对NaCl、干旱胁迫的耐性研究

旨在探讨枣树抗坏血酸过氧化物酶基因ZjAPX在植物渗透胁迫中的作用。将ZjAPX基因转入到模式植物拟南芥,以野生型(WT)、转ZjAPX拟南芥株系T2为试材,进行不同浓度NaCl胁迫和干旱胁迫。结果表明,转基因株系的种子萌发、植株生长均优于野生型株系;荧光定量PCR检测转基因拟南芥植株在干旱和盐胁迫处理10 d后目的基因ZjAPX的表达量显著高于野生拟南芥,表明ZjAPX的高表达明显提高了植株的抗旱和耐盐性。     

外源脯氨酸对盐胁迫下甜瓜脯氨酸代谢的影响

为探明外源脯氨酸对盐胁迫下甜瓜脯氨酸代谢的影响,以甜瓜品种‘雪美’为材料采用营养液栽培,对盐胁迫(100mmol·L-1 NaCl)、盐胁迫下添加外源脯氨酸(100mmol·L-1 NaCl+0.2mmol·L-1 Proline)以及对照3种处理后甜瓜幼苗叶片脯氨酸(Pro)含量、吡咯啉-5-羧酸合成酶(P5CS)、鸟氨酸转氨酶(OAT)和脯氨酸脱氢酶(ProDH)活性进行测定,并对OAT和ProDH基因进行克隆及半定量表达分析。结果显示:与对照相比较,盐胁迫条件下甜瓜幼苗叶片内Pro含量显著增加,P5CS活性增幅大于OAT活性,OAT基因表达量大部分时段内没有增加,ProDH活性下降,ProDH基因表达量减少;盐胁迫下添加外源脯氨酸进一步使幼苗叶片内Pro含量增加、OAT、ProDH活性提高、P5CS活性降低,并且使OAT基因表达量迅速增加、ProDH基因表达量先增加后回落。研究表明,盐胁迫条件下,甜瓜幼苗体内脯氨酸积累主要是通过增强脯氨酸的谷氨酸合成途径和抑制脯氨酸降解来实现;适量外源脯氨酸可以增强盐胁迫幼苗脯氨酸的鸟氨酸合成途径,但对谷氨酸合成途径有一定的抑制作用;通过调节合成和降解2种代谢途径进一步提高了脯氨酸含量,从而增强甜瓜幼苗耐盐胁迫能力。     

转天山雪莲冷调节蛋白基因烟草的获得及其抗寒性鉴定

从已建立的天山雪莲cDNA文库中随机挑取单克隆,采用通用引物M13扩增出基因序列并测序,然后将该序列在GenBank中进行比对,根据该序列设计特异引物进行PCR,最终获得了冷调节蛋白基因siCOR的完整开放读码框(ORF);构建植物表达载体,通过农杆菌介导法获得转基因烟草,用RT-PCR对转化烟草进行鉴定,收取种子后筛选出纯合的T3株系,并通过冷冻胁迫进行抗寒性分析。结果表明:(1)siCOR大小661bp,与禾本科植物粳稻的冷调节蛋白基因一致性最高(65.04%)。(2)与野生型植株相比,转siCOR基因烟草均叶片浓绿、无分支侧芽,生长快;在冷冻胁迫条件下,转siCOR基因的烟草叶片相对含水量、PSⅡ相对量子产率降低幅度、相对电导率和丙二醛含量的升高幅度均低于野生型烟草植株,而且转siCOR基因烟草植株叶片出现萎蔫症状的时间较迟、程度较轻,受到的伤害也较轻。研究发现,在冷冻胁迫条件下,转siCOR基因烟草植株表现出良好的生理和生长优势,显示出了较强的抗寒特征。     

过量表达棉花CBF2基因提高转基因拟南芥抗旱耐盐能力

CBF/DREB是一类植物中特有的转录因子,在植物抵抗逆境胁迫过程中发挥重要功能。本研究从陆地棉(Gossypium hirsutum L.)Coker 312中克隆获得1个棉花CBF/DREB基因,命名为Gh CBF2,该基因编码一个由216个氨基酸组成的CBF蛋白。序列分析结果显示,Gh CBF2与其他植物的CBF蛋白类似,含有AP2转录因子典型的保守结构域。干旱或高盐胁迫处理明显增加了Gh CBF2基因的表达量。亚细胞定位分析结果发现Gh CBF2定位在细胞核中。将Gh CBF2基因构建到由35S启动子调控的植物表达载体p MD上并转化拟南芥(Arabidopsis thaliana L.),结果表明,在干旱和盐胁迫条件下,过量表达Gh CBF2基因拟南芥的成活率显著高于野生型,并且游离脯氨酸和可溶性糖含量也高于野生型,说明转Gh CBF2基因提高了拟南芥的耐盐抗旱能力。采用实时荧光定量PCR方法分析胁迫相关标记基因COR15A、RD29A和ERD6的表达情况,结果显示转基因株系中的表达量显著高于野生型,说明Gh CBF2参与调控拟南芥干旱和盐胁迫相关基因的表达。     

番茄Sly-MIR167的抗冷性研究

以番茄为材料,采用Northern杂交技术,分析番茄MIR167(Sly-MIR167)在低温胁迫下的表达模式,以明确Sly-MIR167在冷胁迫下的分子调控机制,为基因工程在改良番茄品种中的实际应用提供依据。结果显示:(1)25℃下Sly-MIR167在番茄根、茎、花瓣、果实、叶片都有表达,4℃低温胁迫下的表达量均增加,表明Sly-MIR167表达受低温诱导。(2)采用农杆菌侵染构建表达载体并转化番茄获得转基因植株,冷胁迫实验结果显示:转基因植株在冷胁迫处理的生长状况明显优于对照;另外,冷胁迫下2个转基因株系(T2-5和T2-19)的最大光化学效率、叶绿素含量下降幅度明显低于野生型;脯氨酸含量高于野生型;MDA含量低于野生型,表明Sly-MIR167能够提高番茄对冷胁迫的耐受性。(3)通过miRU在线软件预测Sly-MIR167的靶基因为NF-YA1、NF-YA2,利用RT-PCR技术分析其表达下调,证明它们被MIR167负调控。     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

中国黑粉菌属和利罗黑粉菌属

黑粉菌属是Roussel 1806年建立的,全世界记载有三百余种,主要寄生于禾本科,是经济作物及牧草的重要致病菌·长期以来,对黑粉菌的邢子使用过各种名称,如厚垣孢子,冬孢子及黑粉孢子等.本文采用黑粉孢子以区别锈菌的冬孢子. 芳’(1979)在《中国真菌总汇》中列出黑粉菌属五十种及一个变型.作者经过显微结构和超显微结构的研究,承认其中二十九种为正确名称,八种及一变型为异名,顶黑粉菌(Ustilago acrearus Berk.)由于错拼而被废弃.埃地黑粉菌(Ustilago emodensis Berk.)被转移至利罗粉菌属(Liroa).另有十一种黑粉菌因缺少标本留待今后订正.自1979年以后,杨信东(1983)增加黑粉菌属二种我国新纪录,K.范基和郭林(1986)描述一新种,四种新纪录.在本文中,作者描述一新种:鸢尾蒜黑粉(Ustilago ixiolirii Guo L) ,孢子堆生在蒴果内,不开裂,黑色,粉末状.黑粉孢子球形,近球形,稀椭圆形, 12.5-21×10-21μm,黑褐色,壁厚1-1.Sμm,纹饰脑状.是迄今生在石蒜科植物上唯一黑粉菌的种,其它几种黑粉菌均属条黑粉菌属.本文增加七种我国新纪录.共计四十九种,寄生于六科四十四属植物,主要是禾本科和蓼科.这仅是黑粉菌属研究的初步报告,在全国范围内大量采集黑粉菌标本后,作者相信会有更多新种和我国新纪录被发现.利罗黑粉菌属(Liroa)是从黑粉菌属(Ustaligo)分出的,此属为单种属.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.