为探究"夏黑""阳光玫瑰"葡萄在摘心处理后对叶片光合和叶绿素荧光特性的响应,为这两个葡萄品种的栽培研究提供理论依据,以当年生"夏黑""阳光玫瑰"葡萄单株盆栽苗为试验材料,采用顶芽摘心技术进行改善植株光合效率的试验,以不摘心为对照,测定摘心20～21d后标记叶的光合参数和叶绿素荧光参数.结果表明,摘心后,"夏黑""阳光玫瑰"葡萄的净光合速率(Pn)均显著增加,分别比对照增加16.8％、35.7％;气孔导度(Gs)分别比对照增加31.7％、14.2％;胞间CO2浓度(Ci)分别比对照提高8.6％、5.3％;蒸腾速率(Tr)分别比对照提高12.6％、39.8％;摘心后水蒸气压亏缺(VPD)分别比对照降低26.3％、23.6％.摘心有助于提高葡萄叶片的Pn、Gs、Ci、Tr,降低葡萄叶片的VPD.摘心后,两种葡萄叶片的初始荧光(F0)降低,热耗散量子比率(F0/Fm)显著低于对照4.34％、3.66％,而光系统Ⅱ最大光能转换效率(Fv/Fm)分别显著高于对照处理0.93％、0.87％.光合参数Ci、Gs、VPD、Pn、Tr、WUE相互之间存在极显著相关性,叶绿素荧光参数F0、Fm、F0/Fm、Fv/Fm相互存在极显著相关性,但光合参数中只有VPD、Pn、Tr与叶绿素荧光参数显著相关.

为了充分利用广西及南方地区的温光资源,引种适合一年两收的无核、红色、具香味的葡萄新品种,本试验引进郑艳无核葡萄新品种在广西南宁种植,观察其植物学特性、物候期、生长结果习性、果实性状及抗病性等表现,并研究其一年两收栽培技术.经过4年的引种观察,郑艳无核葡萄在广西南宁可成功实现一年两收,表现为红色、无核、早熟、果穗中等、不易裂果、着色均匀、有香气、稳产等特点.4年引种观察期间,一茬果每667 m2产量最多为708.14 kg,二茬果每667 m2产量最多为578.02 kg;一茬果可溶性固形物含量最高为22.97％,二茬果可溶性固形物含量最高为23.71％;一茬果在6月中旬至月末果实完全成熟,二茬果在11月底至12月初果实完全成熟.郑艳无核葡萄在广西南宁引种表现优良,适合在广西南宁及气候类似地区在避雨条件下进行一年两收栽培.

Grapevine is a popular cultivated fruit throughout the world and heat stress is one of the most serious threats to viticulture. However, transcriptional responses, such as molecular properties of photosynthesis and abscisic acid biosynthesis, metabolism and signal transduction pathway of grapevine to heat stress, are still poorly understood. In this study, RNA-seq was carried out for thermostabilized grapevine ‘Kyoho’ leaves. Results showed that 685 and 469 genes were commonly down-regulated and up-regulated at three sampling time-points. The light-dependent reactions of photosynthesis was significantly enriched in up-regulated DEGs at 1 hpt and down-regulated DEGs at R24 hpt. Heat stress impaired the photosynthetic capacity of grapevine leaves, and there was a significant positive relationship between photosynthesis and stomatal conductance at short-term post-heat stress treatment, but the inhibition of HS on Pn was non-stomata limitation for a longer period. Photosystem (PS)Ⅱ was more sensitive to heat stress than PSⅠ, and PsbP, as well as Psb28, played important roles in response to heat stress. The abscisic acid (ABA) content in heat-stress-treated Kyoho plants was higher than that in the control at 1 hpt, but less in heat-stress-treated plants at 4 and R24 hpt, which was regulated by numerous genes involved in the ABA biosynthesis and catabolism pathways. These results help to understand the influence of heat stress on photosynthesis and ABA biosynthesis, metabolism and signal transduction pathway.

试验以桂葡1号×赤霞珠及其343株杂交后代,桂葡1号×Lacrosse及其132株杂交后代作为试验材料,进行白粉抗性鉴定及遗传分析.结果表明:在桂葡1号与赤霞珠组合中,母本桂葡1号的病情指数为39.23,对白粉病表现为感病,父本赤霞珠病情指数为67.83,对白粉病表现为高感;343株F1代杂种群体,有77株高于父本赤霞珠的病情指数,有112株低于母本桂葡1号的病情指数.桂葡1号×Lacrosse组合,双亲对白粉病表现为感病,母本桂葡1号病情指数为39.23,父本病情指数为48.21;在132株F1代杂交种群体中,有42株高于父本Lacrosse的病情指数,有72株株低于母本桂葡1号的病情指数.在2个杂交后代群体中都分离出一定比例的高抗白粉病单株,为开展白粉病抗性葡萄新品种选育提供了材料.

[目的]探究矮壮素处理后'夏黑'葡萄二季果成花的主要生物学过程和重要基因,为促进二季果成花提供理论依据.[方法]以矮壮素处理后'夏黑'葡萄二季果第5～6节位冬芽为试验材料,采用转录组测序技术筛选差异表达基因(DEGs),并对其进行分析.[结果]与对照相比,矮壮素处理后-3 DAF、4 DAF、11 DAF冬芽样品中差异基因数分别为395、557和97个.其中-3 DAF、4 DAF、11 DAF冬芽样品中分别有52、21、39个DEGs上调表达,343、536、58个DEGs下调表达,共有879个DEGs得到注释,富集最显著的Pathway主要有苯丙素和类黄酮生物合成、激素信号转导等.[结论]通过转录组测序和qRT-PCR验证,发现矮壮素处理'夏黑'葡萄后,二季果成花关键节位冬芽内苯丙素生物合成途径中的POD47和PODN1、类黄酮生物合成途径中的STS6、生长素信号转导通路中的IA426、赤霉素信号转导通路中的 GARP6和GA3OX1等基因的表达量发生明显变化,说明矮壮素促进成花的功能可能通过改变树体内激素的含量及比值、次级代谢产物的合成和代谢来实现.

为了探讨摘心与矮壮素(CCC)处理后"夏黑"葡萄枝条内源激素含量变化与枝条生长的关系,以"夏黑"葡萄为试材,在枝条萌芽15d后花序上留2片叶进行摘心处理;矮壮素(CCC)处理为枝条萌芽25d后喷施第1次,间隔8d喷施第2次,2次喷施浓度均为0.5g/L;对照不做任何处理.定期采样分析枝条内源激素含量的变化,同时测量枝条粗度与节间长度.结果表明:摘心处理和矮壮素(CCC)处理能在"夏黑"葡萄枝条生长发育前期促进枝条内脱落酸(ABA)和细胞分裂素(ZRs)的增加,尤其促进ZRs的大量积累,同时促进枝条粗度的增加并减缓枝条节间伸长.生产上可以通过摘心处理及喷施矮壮素控制"夏黑"葡萄的营养生长,从而促进其花芽发育.

[目的]利用酵母单杂交文库筛选巨峰葡萄VvFT基因启动子上游调控因子,挖掘参与调控葡萄开花时间的因子,为揭示花期转变代谢通路和定向改良葡萄品种提供理论参考.[方法]利用PlantCARE分析预测VvFT基因启动子顺式作用元件,并以VvFT基因启动子为诱饵,利用酵母单杂交文库筛选技术,筛选作用在VvFT基因启动子上游的调控因子.[结果]葡萄VvFT基因启动子区域存在13种启动子顺式作用元件,包括A-box、CAAT-box和TATA-box组成型调控元件;光响应调控元件ATC-motif、Box 4、G-Box和GT1-motif;茉莉酸甲酯(MeJA)响应调控元件CGTCA-motif和TGACG-motif;干旱诱导的MYB结合位点元件MBS;玉米蛋白代谢必需的调控元件O2-site;参与生物钟控制的调控元件circadian;厌氧诱导必需调控元件ARE.以VvFT基因启动子为诱饵,筛选获得34条表达序列标签(EST),其中有21条为未知功能,有13条在植物生长、抗逆防御、信号转导、转录调控、蛋白酶等方面均有已知或预测的功能,包括转录抑制因子ft41、GRF1互作因子ft44、NAP1相关蛋白ft64及ft70分子伴侣DnaJ10.酵母单杂交点对点验证结果表明VvDnaJ10和VvFT基因启动子之间有相互作用.[结论]通过酵母单杂交文库筛选获取13条候选EST,虽然大部分EST功能预测分析结果与VvFT调控开花时间无明显的直接关系,但研究结果为进一步探索VvFT转录或表达水平控制葡萄开花时间的分子机制提供侯选基因.

本研究采用同源克隆的方法从玉米自交系昌7-2中克隆获得了一个编码C2结构域蛋白的基因Zmcnp1,该基因具有一个432 bp的开放阅读框,编码143aa的多肽,其中N-端含有一个C2结构域(4～122aa),且存在5个与Ca2+结合的天冬氨酸位点;没有信号肽,属于胞内蛋白;进化上与高粱C2结构域蛋白Sb06g033270进化距离最近.利用RT-qPCR技术对Zmcnp1在玉米不同组织中的表达模式及在不同逆境胁迫下的诱导表达特性进行了研究,发现Zmcnp1主要在玉米根中表达.PEG6000胁迫显著增强了根中Zmcnp1基因的表达;土壤水分胁迫也提高了不同玉米品系B73、Z58和C7-2中Zmcnp1基因的表达,但在Y478中变化不大;盐胁迫(100 mmol/L NaCl)下,玉米根和叶中Zmcnp1在胁迫早期表达下调,后期表达上调.因而,Zmcnp1基因可能参与了PEG6000渗透胁迫、土壤干旱和高盐等非生物胁迫因子的调控.

Bifunctional alginate lyase can efficiently saccharify alginate biomass and prepare functional oligosaccharides of alginate.

Alginate lyases are a group of enzymes that catalyze the depolymerization of alginates into oligosaccharides or monosaccharides. These enzymes have been widely used for a variety of purposes, such as producing bioactive oligosaccharides, controlling the rheological properties of polysaccharides, and performing structural analyses of polysaccharides. The algM4 gene of the marine bacterium Vibrio weizhoudaoensis M0101 encodes an alginate lyase that belongs to the polysaccharide lyase family 7 (PL7). In this study, the kinetic constants Vmax (maximum reaction rate) and Km (Michaelis constant) of AlgM4 activity were determined as 2.75 nmol/s and 2.72 mg/mL, respectively. The optimum temperature for AlgM4 activity was 30 °C, and at 70 °C, AlgM4 activity dropped to 11% of the maximum observed activity. The optimum pH for AlgM4 activity was 8.5, and AlgM4 was completely inactive at pH 11. The addition of 1 mol/L NaCl resulted in a more than sevenfold increase in the relative activity of AlgM4. The secondary structure of AlgM4 was altered in the presence of NaCl, which caused the α-helical content to decrease from 12.4 to 10.8% and the β-sheet content to decrease by 1.7%. In addition, NaCl enhanced the thermal stability of AlgM4 and increased the midpoint of thermal denaturation (Tm) by 4.9 °C. AlgM4 exhibited an ability to degrade sodium alginate, poly-mannuronic acid (polyM), and poly-guluronic acid (polyG), resulting in the production of oligosaccharides with a degree of polymerization (DP) of 2–9. AlgM4 possessed broader substrate, indicating that it is a bifunctional alginate lyase. Thus, AlgM4 is a novel salt-activated and bifunctional alginate lyase of the PL7 family with endolytic activity.

[Objective] A marine Vibrio X511 with strong ability to use alginate was isolated from the rotten Sargassum in the Weizhou Island of the North Sea.The metabolic pathway of using alginate by Vibrio X511 was studied by transcriptome sequencing.[Methods] Transcriptions sequencing was done by Illumina Hi Seq2500 sequencing platform and the transcriptome of the strains cultured in alginate and glucose were sequenced.Differential transcripts were compared and analyzed, and the sequencing results were confirmed by fluorescence quantitative PCR.The fluorescence quantitative PCR was applied to verify the sequencing results.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes were used to annotate the function and pathway in differential transcripts.[Results] There were 2024 differentially expressed genes in the culture of alginate compared to those in glucose, with 1066 genes up-regulated and 958 genes down-regulated.Some genes in the ubiquitous metabolic pathway were also differentially expressed in different cultures.The transcriptions of all the genes involved in alginate utilization and the key genes of synthetic ethanol in Vibrio X511 were up-regulated.In addition, the strain had a unique way to utilize alginate and one of the metabolic processes has not been reported in Vibrio.[Conclusion] Our findings enrich the research of biological method for degradation of alginate, and provide valuable data for the research of large seaweed biomass-based energy.

[Objective]Screening thermostable α-amylase and expressed in Escherichia coli,and analyzing the recombinant enzyme characterization.[Methods]The strain Anoxybacillus sp.GXS-3 is isolated from soil samples collected from Tengchong hot spring,with M9 medium and soluble starch as the sole carbon source.According to the amino acid conserved sequence of amylase,degenerate primers are designed for PCR amplification.and then the target sequence is amplified and amplified to obtain the amylase gene AmyGX.AmyGX is ligated with the expression vector pQE30, introduced into E.coli M15, and the recombinantα-amylase is isolated and purified by NTA-affinity chromatography and its enzymological properties are analyzed.[Results]The gene of AmyGX is 1 515 bp in length and encoded a protein of 505 amino acids,of which,the first 23 amino acid residues are signal peptide sequences.The molecular mass of the recombinant AmyGX is 58.04 kDa.Assayed with soluble starch as substrate,this recombinant enzyme displays optimal activity at pH 8.0 and 60℃ with an apparent Km value of 3.14 mg/ml and Vmax of 0.19 U/mg respectively.The temperature at which 50% of the enzyme activity is lost after 30 min heat treatment (T3050) is 65.2℃.Zn2+、Cu2+、Co2+、Fe3+ 、Ba2+has significantly inhibitory effect on the activity of the recombinant enzyme,whereas Na+、K+could increase the activity and Mg2+、Ca2+ has slightly effect on it.[Conclusion]AmyGX is a moderate enzyme,it could be applied in the field of paper,detergent production and the removal of waste material in environment.

The alginate lyase gene in marine Vibrio sp.X511 was expressed by Escherichia coli to realize the large-scale preparation of alginate lyase and research enzymatic properties.By analyzing the sequence,the first 20 amino acids of the enzyme were predicted as the signal peptide,the protein molecular mass without the signal peptide was 30 254.28 Da,the isoelectric point was 8.75,the molecular formula was C1368H2100N364O401S6,and the corresponding gene sequence was named as alg1987.The construction program of bacteria recombination was as follows:the specific nucleic acid primers were designed and the alg1987 was obtained by PCR;the recombinant plasmid pET-30(a)-alg1987 was introduced into E.coli Trans5t for sequencing;the positive plasmid pET-30(a)-alg1987 was extracted and introduced into E.coli BL21,then expressed by the induce of IPTG.The supematant of recombinant bacteria cytosol was induced and detected by SDS-PAGE,it was found that the molecular mass of the alginate lyase by heterologous expression was similar to the predicted value.The expression conditions of alginate lyase in recombinant bacteria were optimized.The results showed that optimum parameters were as follows:induction temperature 16 ℃,time 16 h,induction agent IPTG concentration 0.8 mmol/L.

采用响应面法优化海洋弧菌X511的产酶发酵培养基,提高其胞内褐藻胶裂解酶产量.通过单因素试验研究了不同碳源、不同氮源、海藻酸钠、氯化钠、蛋白胨、硫酸亚铁、硫酸镁和磷酸氢二钾对菌株产胞内酶活力的影响,在此基础上,利用Plackett-burman试验确定海藻酸钠、氯化钠、蛋白胨和硫酸镁对胞内酶产量的影响.通过响应面试验构建回归方程,结果表明,最佳发酵培养基成分为海藻酸钠9.0 g/L,NaC1 31.6 g/L,蛋白胨15.0g/L,MgSO4·7H2O 1.5 g/L.在此条件下,该菌株的胞内褐藻胶裂解酶的活力为(20.65±0.14) U/mL,较优化前的酶活提高了64.4％.

Objective]Alginate lyase in Shewanella haliotis BP-1 strains was studied illustrate its biological activity of degrading alginate.[Methods]The gene cloning technology and the Escherichia coli heterologous expression technology were applied to overexpress the alginate lyase;And the enzyme activity was analyzed after the crude enzyme was separated and purified by DEAE Sepharose FF chromatogra-phy.[Results]The alginate lyase gene Alg 1 7S , with a size of 2 1 5 7 bp,was cloned from S. haliotis BP-1 strain genomic DNA and encoded an alginate lyase Alg17S,which belonged to pol-ysaccharide lyase(PL)1 7 family and had a size of 79 726 Da protein(including an N-terminal signal peptide of 26 amino acid signal peptide).Alg17S showed high sequence identity of 5 2% with PL-17 protein sequence Alg17C from Saccharophagus degradans 2-40.Both the purified recombi-nase Alg17S and the △snAlg17S(without the N-terminal signal peptide of 26 amino acids)can degrade alginate,but the enzymatic activity of △snAlg17S revealed a specific activity of 9 635 U/mg,which was more efficient than Alg17S.[Conclusion]The recombinant alginate lyase △s-nAlg17S that has both high-level expression and high enzymatic activity could be a potential en-zyme for further researching on the alginate saccharification and the biofuels production.

Abtract:[Objective]The aim of this study is to analyze the interaction between the different groups ofβ-carotene in the heating process.[Methods]Two-dimensional correlation spectrosco-py was applied to study the dynamic spectral changes ofβ-carotene from 30℃ to 100℃.[Re-sults]The changes of absorption characteristic peaks ofβ-carotene were inconspicuous in the conventional FTIR spectra and second derivative FTIR spectra during 30℃ to 100℃,which in-dicated that they had no oxidation reation.Two-dimensional correlation analysis showed that the changes of absorption peaks at 968 cm-1 ,1 442 cm-1 ,2 9 6 6 cm-1 and 3 0 1 2 cm-1 were more sen-sitive to temperature.Meanwhile,the order of different groups changes induced by temperature were as follows:the spectral changes of methyl-ene were faster than methyl,the spectral changes of methyl C-H symmetric stretching vibration in low wavenumber were faster than methyl anti-symmetric stretching vibration in high wavenumber,and olefin hydrocarbon symmetric stretching vibration were prior to olefin hydrocarbon anti-symmetric stretching vibration.[Con-clusion]This provides experimental basis for the mechanism of the conformational change ofβ-carotene in heating process.

【Objective】The apoptosis of yeast cells was induced by ultraviolet and its process was studied in order to understand the changes of biological macromolecules and the molecular mechanism.【Methods】Laser tweezers Raman spectroscopy(LTRS)was used to monitor the dynamics of the intracellular biological macromolecules in real-time during the apoptosis process of yeast cells stressed with UV at both cellular population level and single-cell level,respectively.【Results】Lethal doses of UV irradiation could cause cell apoptosis.The intensities ofRaman peaks,which were assigned to nucleic acids(1085cm-1,1300cm-1),proteins(850cm-1,1440cm-1,1604cm-1,1655cm-1)and lipids(1085cm-1,1300cm-1,1440cm-1),decreased significantly as a function of the duration of cell apoptosis at cellular population level,suggesting that the content of nucleic acids,proteins and lipids reduced gradually during the time that the yeast cells were undergoing apoptosis induced by UV.The peak of 1604cm-1,which was called the Raman spectroscopic signature of life in yeasts,was a marker of Raman band for cell metabolic activity.Its intensity had the sharpest decline by 60%at the late stage of apoptosis,implying that the energy metabolism and breathing capacity were decreased during the process of cell apoptosis,which related to the changes of ergosterol structure and function theoretically.However,the changes of the intensities of Raman peaks at 850cm-1,1085cm-1,1300cm-1,1440cm-1 and 1665cm-1 between the group cells and the single cells were different significantly in the period of 90~120min and 125~167min,demonstrating that the heterogeneities of the single cells were masked by the average spectroscopy of the population cells.【Conclusion】LTRS can be used to directly and truthfully detect the kinetics of apoptotic process of yeast cells under UV irradiation at the single cell level and probe cellular heterogeneity.