普通小麦TaADF8基因的克隆及表达分析

肌动蛋白解聚合因子(actin-depolymerizing factor,ADF)普遍存在于真核细胞中,为低分子量的肌动蛋白结合蛋白,在调控细胞内肌动蛋白纤维的聚合和解聚中起关键作用。为给深入研究TaADF8基因在小麦中的功能机理奠定基础,并为进一步丰富小麦ADF基因研究内容提供理论参考,本研究利用电子克隆策略从小麦品种CP53中克隆出TaADF8基因(GenBank登录号为KJ864962)后对其进行序列分析,并进一步采用荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术对其在小麦不同组织间的表达差异及不同非生物胁迫下的表达模式进行分析。核酸序列分析表明,该基因全长695bp,拥有完整的ORF,编码142个氨基酸。氨基酸序列分析表明,该蛋白含有保守的ADF同源区和PIP2结合结构域,且在氨基端有核定位信号。进化和聚类分析表明,小麦TaADF8基因与大麦HvADF2基因、HvADF3基因和水稻OsADF3基因亲缘关系较近,蛋白相似度分别为75.35%、93.66%和67.86%。qRT-PCR表达特性分析显示,该基因为组成型表达,在根、茎、叶、颖壳和雄蕊中均表达,且在根、叶和雄蕊中表达量较高;该基因表达受低温的强烈诱导,同时也受水分、高盐和外源脱落酸胁迫诱导。     

巴西橡胶树ADF6基因的克隆与表达分析

肌动蛋白解聚因子(Actin Depolymerizing Factor,ADF)是一种重要的肌动蛋白结合蛋白。ADF通过参与调控肌动蛋白的装配和解聚在多种生理过程中发挥重要作用。本研究通过RT-PCR技术从橡胶树胶乳中获得HbADF6基因的cDNA和基因组序列,序列分析显示HbADF6基因的开放阅读框为441 bp,共编码146个氨基酸,基因组序列由2个内含子和3个外显子组成。实时荧光定量PCR结果表明,HbADF6基因在所有检测组织中均表达,其中树皮中表达量最高,叶片中表达量最低。HbADF6基因的表达受乙烯利(ET)和碘化钾(KI)调控,并与排胶时间和死皮有关。此结果表明HbADF6基因可能在橡胶树乙烯响应、死皮和排胶过程中发挥重要作用。      

玉米脂肪酸α-双加氧酶cDNA克隆与表达分析

以玉米自交系丹340为实验材料,同源克隆到玉米脂肪酸α-双加氧酶基因(ZmDOX)cDNA序列,并利用半定量与定量RT-PCR分析其在不同组织和PEG胁迫下的表达特征。序列分析结果表明,该cDNA序列包含1 857 bp完整的开放阅读框,编码619个氨基酸残基的蛋白产物。玉米、水稻、小麦中DOX氨基酸序列高度同源,聚集在同一进化枝,而双子叶植物存在两类DOX蛋白。组织表达特征分析发现,ZmDOX在根、茎、叶中均有表达,其中在根中表达量最高。PEG胁迫条件下,根中ZmDOX对胁迫的响应较快且增加幅度较大,而在叶中表达量有降低的趋势。     

木薯MeHDS2基因的克隆与分析

采用RT-PCR技术,从木薯SC124 cDNA中克隆得到一个具有完整阅读框的基因,命名为MeHDS2,该基因全长2 529 bp,编码842个氨基酸。蛋白质保守结构域分析结果表明,MeHDS2蛋白含有保守的1个HD结构域、1个b-zip结构域、1个START结构域和1个MEKHLA结构域;蛋白质空间结构预测显示其具有3个典型的α螺旋结构;亚细胞定位结果表明,MeHDS2蛋白在植物细胞核中特异表达,因此,推测其为HD-ZIP转录因子家族中的一员。基因表达分析结果表明,MeHDS2基因在木薯根、茎、叶中都有表达,但表达丰度不同。木薯干旱处理14 d后,自形态学顶端往下数第三、四片叶片的MeHDS2表达量显著高于根、茎及其他位置叶片组织。本研究为木薯抗旱分子育种提供了理论基础。     

小麦细胞色素P450(TaP450)基因的克隆与序列分析

细胞色素P450是一种多功能氧化酶,在植物体内担当着生物合成、代谢解毒以及抗逆等重要功能。本研究采用RACE方法从普通小麦中同源克隆到一个新的P450基因,命名为TaP450(Genbank No.KJ541960)。该基因基因组序列全长为2 643bp,含有2个外显子和1个内含子,cDNA全长为2 033bp,含有一个1 557bp的开放读码框,推导蛋白含518个氨基酸;序列分析发现其具有保守的P450结构域,但该蛋白与已报道的小麦P450蛋白序列有较大差异,表明它是小麦P450家族的新成员;蛋白结构特征分析发现,该蛋白的分子量为57.092kD,等电点为8.63,N端具有一个含29个氨基酸的跨膜结构和一个含22个氨基酸的信号肽,亚细胞定位分析表明该蛋白属于分泌蛋白。其在小麦叶片、茎、根与种子中均有表达,但在叶片和茎中表达量最高;在胁迫处理下,该基因的表达量均上调,且在盐胁迫处理下上调尤为显著,表明该基因与盐胁迫密切相关。     

蝴蝶兰蔗糖合成酶基因的cDNA克隆及其表达分析

利用低夜温诱导的蝴蝶兰花芽的抑制消减杂交文库中分离的蔗糖合成酶基因的ESTs片段,采用RT-PCR和RACE技术,从蝴蝶兰花芽中克隆得到蔗糖合成酶基因的全长cDNA,命名为PhSUS,GenBank登录号JX162557.该基因全长2 816 bp,包含147 bp的5’非编码区、218 bp的3 '非编码区和一个长度为2 451 bp编码816个氨基酸的开放阅读框.PhSUS预测的分子量为93.30 ku,等电点为5.95.蛋白同源性分析结果表明,PhSUS与兰科植物的文心兰、石斛兰和莫氏兰(Mokara)等的蔗糖合成酶有很高同源性.保守结构域分析结果表明,PhSUS含有蔗糖合成酶和葡糖基转移酶两个保守功能域及4个ADP结合位点.表达分析结果表明,PhSUS在检测的组织中都有表达,但表达丰度不同.PhSUS在花蕾发育中后期、成熟花和花器官的表达量都较营养阶段根和叶中的表达量高.PhSUS的克隆为研究其参与花芽分化和发育的表达调控奠定了重要基础.     

野生大豆(Glycine soja)C2H2型锌指蛋白基因的克隆与序列分析

根据大豆SCOF-1的CDs区设计特异引物,通过PCR和RT-PCR从野生大豆01-197中克隆到含两个锌指的C2H2型锌指蛋白基因,命名为GjC2H2,GenBank登录号为FJ172776.对其进行核苷酸和氨基酸同源性分析及系统发生分析,结果表明:该基因内部无内含子,长度为702bp,分子量为25.13KD,与其他锌指蛋白基因有很高的同源性,推测其与植物抗逆性有关.     

野生大豆转录因子GsWRKY57基因的克隆与抗旱性功能分析

WRKY转录因子是近年来在植物中发现的一类重要转录因子,在植物抗逆相关过程中发挥重要作用。本研究通过对野生大豆转录组数据进行分析,从野生大豆中克隆得到GsWRKY57基因的CDS序列。该基因开放阅读框为903bp,编码300个氨基酸残基,分子量为34.23kD,等电点为5.88。氨基酸序列分析显示该蛋白含有一个WRKY保守结构域,属于III类WRKY转录因子。系统发育树分析表明该蛋白与栽培大豆同源性最高、其次是赤豆、木豆。启动子作用元件分析预测显示该基因可能存在多种非生物胁迫作用元件。组织特异性表达分析表明该基因在大豆叶片中高丰度表达,然后依次是茎、花、荚、根。GsWRKY57基因表达受茉莉酸、水杨酸、脱落酸、干旱等植物逆境诱导。过表达GsWRKY57基因的转基因拟南芥植株相对于野生型耐旱能力增强。     

番荔枝中一个SWEET家族基因的克隆与表达分析

以番荔枝不同组织样品为材料,通过基因文库筛选,利用RT-PCR技术克隆出1个1227 bp的基因,命名为AT-SWEET16-1,该基因编码408个氨基酸,该氨基酸序列在N端以α螺旋形成THB结构域。生物信息学分析结果表明,该蛋白分子量为44.8 kDa,等电点为8.87。进化分析结果发现其与海枣（Phoenix dactylifera）相类聚。qRT-PCR分析结果表明,该基因在植株的根、茎、嫩叶、老叶、花蕾、花苞、幼果、成熟果中均有表达,AT-SWEET16-1基因在不同组织中的表达量依次是：成熟果>茎>根>花蕾>幼果>老叶>嫩叶;该基因在不同果实发育阶段中的表达情况为：在果实不同发育阶段,果柄中的表达量最高,果肉、果皮中则相对较低,种子中最低;但果实成熟期该基因在果柄、果肉中的表达量最高。原位杂交实验观察发现,基因表达位置为果柄韧皮部、果肉细胞膜间,结合基因表达分析结果,预示该基因在植株糖分积累与转运等方面起到一定的作用。     

橡胶树胚胎晚期丰富蛋白基因HbLEA1的克隆和表达分析

胚胎晚期丰富蛋白（LEA）是与植物抗逆反应相关的一类重要蛋白。本研究克隆了橡胶树LEA家族一个新基因的全长cDNA序列,命名为HbLEA1。该cDNA全长1 183 bp,其中5'UTR区60 bp,3'UTR区154 bp,CDS区969 bp,共编码322个氨基酸,分子量大小为36 ku,等电点4.84,含有LEA-2和WHy（Water Stress and Hypersensitive response）结构域,属于LEA_2类LEA蛋白。组织表达分析结果表明,HbLEA1在种子中表达量最高,其次是胶乳和稳定叶,在芽中的表达量最低。在未开割树的胶乳中,机械伤害和割胶处理均明显下调HbLEA1表达。在橡胶树幼苗中,低温胁迫处理后该基因在叶片、树皮和根中的表达呈上升趋势,而在高温和干旱胁迫中其表达变化并不明显。结果显示,HbLEA1基因可能参与橡胶树的胁迫应答和胶乳代谢调控。     

香蕉Actin1启动子的分离及启动活性的分析

从香蕉基因组DNA中分离香蕉Actin1启动子序列，序列测定结果表明，该片段1253bp，包含完整的G-box和TATA box，5’非翻译区和其下游的内含子，与文献报道序列的同源性达97．53％。以全长序列取代植物表达载体pCAMBIA3300上的35s启动子序列构建成瞬间表达载体pCAMBIA—Act1，以内含子序列Hind Ⅲ和XbaⅠ双酶切片段（584bp）取代植物表达载体pBI121上的35 S启动子，构建成瞬间表达载体pBI121-Act1-1。采用基因枪法对香蕉根、叶和果实的薄片进行转化，转化材料经培养3h，采用分光光度法测定各组织GUS的活性。结果表明，Actin1启动子在所转化的3种组织中均可启动报告基因的表达，表达活性与35 S启动子接近，具有组成型表达的特点。内含子部分序列（584bp）也具有启动活性，但启动活性较低。     

Nitrogen in the detergent fibre fractions of temperate legumes and grasses†

Data are few on concentrations of nitrogen (N) in the cell wall and lignocellulose (neutral- and acid-detergent fibre (NDF and ADF), respectively) of herbage. Herbage N can be partitioned into neutral- and acid-detergent soluble and insoluble N to crudely estimate rapidly degradable (N soluble in neutral detergent), slowly degradable (neutral-detergent fibre N (NDFN) minus acid-detergent fibre N (ADFN), herein termed available fibre N (AFN)), and indigestible N (ADFN) in the rumen. Our objective was to examine the effects of herbage species, maturity stage, and plant part on N in the NDF and ADF of lucerne (Medicago sativa L.), red clover (Trifolium pratense L.), timothy (Phleum pratense L.), and smooth bromegrass (Bromus inermis Leyss.). Herbage was harvested at four 2-week intervals during spring of 1984 and 1985. Leaves, stems and total herbage were analysed for total N, and N in NDF and ADF. Concentrations of N in ADF in leaves, stems, and total herbage of lucerne and clover were twice that of grass ADF. This may be due to greater lignin concentrations generally reported in legumes. Concentrations of NDFN in leaves of lucerne, clover, timothy, and bromegrass were 9–8, 26.4, 8.6, and 6.4 g kg^?1 of NDF, respectively, averaged over harvests and years, whereas stem concentrations were 4.2, 4.6, 32, and 2.4 g kg^?1 of NDF, respectively. Leaf concentrations of ADFN in lucerne, clover, timothy, and bromegrass were 4.6, 5.3, 20, and 1.6 g kg^?1 of ADF, respectively, averaged over harvests and years, whereas stem concentrations were 3.4, 33, 14, and 14 g kg-’of ADF, respectively. As a proportion of the total N, NDFN and ADFN increased with plant maturity in leaves, stems, and herbage. In stems, 49% of NDFN was ADFN, whereas in leaves only 21% of the NDFN was ADFN. The larger portion of ADFN in stems probably reflects the larger proportion of lignified xylem and other structural tissues, which have a greater proportion of lignified secondary cell walls, whereas the greater proportion of NDFN in leaves may mirror the greater amount of mesophyll cells with primary cell walls. More than 80% of the total N in leaves, stems, and herbage was in the cell solubles, which may be rapidly ruminally degradable.     

花生肌动蛋白基因的克隆及序列分析

以花生(Arachis hypogaeaL.)为材料,研究肌动蛋白(actin)基因序列。分离了高纯度RNA。以RNA为模板,以RT-PCR方法,克隆了花生actin基因cDNA序列,长度为384 bp,编码128个氨基酸残基。此cDNA序列(命名为Ahactin)与几种高等植物actin氨基酸序列相似性大于80%。     

Nitrogen concentrations in the cell wall and lignocellulose of smooth homegrass herbage*

Applying nitrogen (N) fertilizer to grass generally increases N concentration in herbage. Not as well documented, however, are N fertilizer effects on concentrations of N in plant cell walls and lignocellulose. The objective four study was to ascertain how N fertilizer applied with or without a nitrification inhibitor would affect N concentrations in the cell wall (neutral-detergent fibre (NDF)) and lignocellulose (acid-detergent fibre (ADF)) of smooth bromegrass (Bromus inermis Leyss). Nitrogen fertilizer ((NH₄)₂SO₄) was applied at 0, 125, or 230 kg N ha^?1 in combination with 0, 2–5, or 50 kg ha^?1 of nitrapyrin (2- chloro-6-(trichloromethyl) pyridine) to smooth bromegrass in April of 1985 and 1986. Herbage was harvested at 2, 4, 6 and 8 weeks after treatment. Freeze-dried herbage samples were analysed for in vitro digestible dry matter (IVDDM), total N, and N in neutral- and acid-detergent fibre (NDFN and ADFN, expressed as g kg^?1 NDF and ADF, respectively). Nitrapyrin had no effect on IVDDM, total N, NDFN or ADFN, Nitrogen fertilizer increased concentrations of total N in herbage dry matter, and increased N concentrations in NDF and ADF. Most of the increase in total N concentration occurred in the neutral-detergent soluble portion. When NDFN and ADFN concentrations were expressed on a total N basis (g k g^?1 of total N), N fertilizer had no effect. Available fibre N (AFN; calculated as NDFN minus ADFN, each expressed as g kg^?1 total N) was not affected by N fertilizer. The average AFN concentration was 93 g kg^?1 total N. Nitrogen fertilizer did not affect herbage quality; however, age of herbage had marked effects on herbage quality.     

Ensiling peas, ryegrass and wheat with additives of lactic acid bacteria (LAB) and cell wall degrading enzymes

The effect of applying a commercial inoculum (at 1.5 × 10⁵ cfu g^-1) containing Streptococcus faecium (Lacticil M74®. Medipharm, Sweden), and cell wall degrading enzymes (Celluclast® and Viscozyme®, Novo, Denmark) to pea, ryegrass and wheat silages was studied under laboratory conditions. Celluclast was applied at 0.03 Novo Cellulose Units (NCU) and Viscozyme at 0.024 Fungal β-glucanase (FBG) per g fresh crop. The peas and wheat used were at three stages of maturity, and the ryegrass used was either direct-cut or wilted. This yielded a wide range of dry matter (DM) and fibre content. Inoculum use improved the ensiling process of most of the forages tested, as indicated by a faster and greater decrease in pH, and by a faster and larger build-up of lactic acid. Inoculum, however, lowered the aerobic stability of wheat silages. The addition of the enzymes alone did not affect the ensiling process. In silages of peas and wheat at the flowering stage, the combination of inoculum and enzymes resulted in a improvement to the ensiling process, as compared with inoculum only. The combined treatment of inoculum and enzymes resulted in a lower percentage of neutral detergent fibre (NDF) and acid detergent fibre (ADF) in the silages of the flowering peas and of the direct-cut ryegrass. The NDF and ADF contents in the inoculum plus enzyme-treated pea silages made at the flowering stage were 322 and 296 g kg^-1, compared with 365 and 327 g kg^-1 in the control silages. The NDF and ADF contents in the silages made from the direct-cut ryegrass were 312 and 205 (treated with inoculum plus enzymes) vs 383 and 257 g kg^-1 (control). However, the DM rumen digestibility of the silages was not improved by any of the treatments. It is concluded that a suitable inoculum could be beneficial to the ensiling process. The benefit expected from the application of cell wall degrading enzymes to silages is as yet unclear.     

优质青贮玉米自交系筛选与评价

以448份玉米自交系为材料,对玉米全株的品质含量指标蛋白、淀粉、中性洗涤纤维(NDF)及酸性洗涤纤维(ADF)进行评价,筛选高蛋白/淀粉、低纤维的青贮玉米自交系。结果表明,在所有供试材料中,蛋白含量变化不大,淀粉含量与纤维(NDF、ADF)含量极显著负相关。以淀粉、NDF和ADF品质含量为指标,通过聚类分析,筛选出7份高淀粉(47.50%~51.20%)、低纤维(ADF为9.08%~12.93%,NDF为26.67%~31.07%)含量的青贮玉米自交系,分别为P124-3、P125-12、P128-1、P132、13634-3、12099-2和12906-1。     

The accuracy of using the ratio of nitrogen to acid detergent fibre in faeces to partition nitrogen between faeces and urine in total excreta collections from ewes

A comparison was made of the estimates of the outputs of nitrogen (N) in faeces and urine on Awassi ewes using two methods: (1) conventional collection in digestibility crates, with urine acidified to prevent volatilization (separation method); (2) collection of both faeces and urine together in bags placed over the tail of the ewe and using the ratio between N and acid detergent fibre (ADF) in samples taken from the rectum to partition the total N output between faeces and urine (N:ADF method). The comparison was made on eight ewes in a crossover design using three collection periods of 5 d. There were no significant differences between the methods in the estimates of nitrogen in urine and faeces, although differences between methods in the estimation of total N output were close to being significant. The precision of the estimates was lower with the N:ADF method.     

四大玉米杂优类群自交系的全株饲用营养价值分析

对我国现行的四大玉米杂优类群中25个自交系的全株玉米营养价值进行了初步测定,结果表明:在四大玉米杂优类群中,瑞德群的粗蛋白(CP)含量最低,粗脂肪(CF)含量最高,中性洗涤纤维(NDF)和酸性洗涤纤维(ADF)含量居中;兰卡斯特群的ADF含量比其它类群低;塘四平头类群的CF含量最低,NDF和ADF含量却最高;旅大红骨群的NDF含量最低,CP含量最高。单株鲜重、单株干重、粗蛋白、粗脂肪和中性洗涤纤维含量在四大类群之间无显著性差异。旅大红骨群与塘四平头群之间的NDF含量有显著差异;ADF含量在兰卡斯特群和瑞德群、塘四平头群和旅大红骨群之间存在着显著性差异。