通过鉴定猕猴桃多胺氧化酶(PAO)基因家族成员,分析其在猕猴桃果实生长发育、成熟及储藏过程中的表达变化,发掘与猕猴桃果实成熟、软化相关的重要候选基因,为调控猕猴桃鲜果的发育及采后保鲜提供参考.以拟南芥AtPAO、水稻OsPAO蛋白序列作为参考序列,利用BLAST程序分别从中华猕猴桃、毛花猕猴桃、软枣猕猴桃基因组或转录组中筛选和鉴定PAO家族成员;利用Compute pI/Mw分析蛋白质分子质量、理论等电点,利用SignalP-4.1、TMHMM-2.0预测信号肽和跨膜区,利用WoLF PSORT进行亚细胞定位预测;利用MEGA 6构建系统发育进化树,利用MEME进行保守基序分析,利用TBtools分析PAO基因的共线性关系;基于转录组数据和荧光定量PCR技术,分析PAO基因的组织表达情况及其在猕猴桃果实生长发育、成熟及储藏过程中的表达变化.结果表明,中华猕猴桃、毛花猕猴桃、软枣猕猴桃中各存在4、4、5个PAO基因,分别是AcPAO1-4、AePAO1-4、AaPAO1-5.AcPAO、AePAO位于中华猕猴桃、毛花猕猴桃的8条染色体上,基因长度为1 722～9 837 bp.AcPAO、AePAO、AaPAO编码的氨基酸数目为420～496个,分子质量为45.74～55.92 ku,等电点为5.29～5.79,均不含有信号肽和跨膜区;AcPAO、AaPAO主要定位在过氧化物酶体,而AePAO分布范围较广;依据系统进化关系,13个猕猴桃PAO蛋白分属于3个类群,同一类群含有相似的motif分布及保守结构域;AcPAO、AePAO间存在4对共线性基因,它们在进化中经历了纯化选择.AcPAO3的表达量随猕猴桃果实成熟、软化呈现先升高后降低趋势,且能够响应不同储藏温度;AaPAO为组成型表达,多数成员的表达量在果实储藏过程中亦呈现先升高后降低的趋势.

羟基肉桂酰辅酶A:莽草酸/奎宁酸羟基肉桂酰转移酶(HCT)是木质素合成过程中的关键酶之一.本研究构建了菊芋HtHCT基因的植物表达载体pCAMBIA1390-Ht HCT,分别对拟南芥和本氏烟进行遗传转化,并对转基因植株进行生理生化指标测定.PCR和RT-PCR结果表明,HtHCT基因已经插入到受体植物基因组中,并能进行转录;转基因植株生理生化指标检测表明,HtHCT基因能够调节拟南芥和本氏烟中木质素的含量及组成,并对受体材料的类黄酮合成亦有一定的影响.

为探明新疆不同核桃资源的蛋白质与氨基酸含量之间的关系,采用凯氏定氮法和高效液相色谱法测定了26份新疆核桃种仁的蛋白质和氨基酸含量,采用相关性分析和通径分析方法分析了蛋白质与氨基酸的相关性.结果 表明:26份新疆核桃种仁中以'扎74'的蛋白质含量和总氨基酸含量最高,分别为19.70、16.92 g/100 g;相关性分析显示甘氨酸、精氨酸等13种氨基酸含量均与蛋白质含量呈极显著正相关,以甘氨酸相关系数最大,为0.906;通径分析结果显示天冬氨酸和谷氨酸对蛋白质含量的影响较大,通径系数分别为4.447和4.421.因此,在核桃育种中可通过采取提高天冬氨酸和谷氨酸含量的策略增加核桃蛋白质含量.

为揭示菊芋(H elianthus tuberosus L.)查尔酮合成酶(chalcone synthase,CHS)基因的功能,以菊芋'廊芋8号'叶片为材料,采用同源克隆和RT-PCR技术,克隆得到菊芋 CHS基因命名为 HtCHS,其编码区全长为1 197 bp(GenBank登录号M N124515),编码398个氨基酸.H tC H S的基因组DNA(gDNA)全长为1 567 bp,含2个外显子和1个内含子.序列比对和系统进化树分析显示,不同植物中的CHS氨基酸序列具有极高的同源性,菊芋与同属的向日葵CHS氨基酸序列一致性达到99.25％,共聚于一个分支.HtCHS蛋白相对分子质量为43.61 ku,等电点为6.33.HtCHS蛋白具有查尔酮合成酶结构域,属于查尔酮合成酶超家族成员,HtCHS不含信号肽和跨膜区,属于非分泌蛋白.实时荧光定量PCR结果显示,H tCH S在根中表达量最高.与对照相比,150 mmol·L-1 NaCl和20％PEG 6000处理6 h时 H tC H S表达显著上调,处理12 h时显著下调.原核诱导表达分析结果显示,成功诱导出与预测蛋白大小一致的目的蛋白.

BACKGROUND:The H+-PPase (pyrophosphatase) gene family is an important class of proton transporters that play key roles in plant development and stress resistance. Although the physiological and biochemical functions of H+-PPases are well characterized, the structural evolution and functional differentiation of this gene family remain unclear.RESULTS:We identified 124 H+-PPase members from 27 plant species using complete genomic data obtained from algae to angiosperms. We found that all analyzed plants carried H+-PPase genes, and members were not limited to the two main types (type I and II). Differentiation of this gene family occurred early in evolutionary history, probably prior to the emergence of algae. The type I and II H+-PPase genes were retained during the subsequent evolution of higher plants, and their copy numbers increased rapidly in some angiosperms following whole-genome duplication (WGD) events, with obvious expression pattern differentiation among the new copies. We found significant functional divergence between type I and II H+-PPase genes, with both showing evidence for positive selection pressure. We classified angiosperm type I H+-PPases into subtypes Ia and non-Ia, which probably differentiated at an early stage of angiosperm evolution. Compared with non-Ia subtype, the Ia subtype appears to confer some advantage in angiosperms, as it is highly conserved and abundantly expressed, but shows no evidence for positive selection.CONCLUSIONS:We hypothesized that there were many types of H+-PPase genes in the plant ancestral genome, and that different plant groups retained different types of these genes. In the early stages of angiosperm evolution, the type I H+-PPase genes differentiated into various subtypes. In addition, the expression pattern varied not only among genes of different types or subtypes, but also among copies of the same subtype. Based on the expression patterns and copy numbers of H+-PPase genes in higher plants, we propose two possible evolutionary trajectories for this gene family.

羟基肉桂酰辅酶A:莽草酸/奎宁酸羟基肉桂酰转移酶(hydroxycirmamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase,HCT)在植物本质素合成过程中具有重要作用.本研究以菊芋‘廊芋8号’为材料,成功克隆到一个菊芋HCT基因——HtHCT,其不含内含子,开放阅读框(ORF)为1 293 bp,编码430个氨基酸.HtHCT含有HCT蛋白共有的保守序列HXXD和DFGWG.系统进化分析表明,HtHCT与向日葵HCT蛋白(QBM78942.1)亲缘关系最近,共聚于一支.原核表达结果表明,HtHCT蛋白成功被诱导表达.荧光定量PCR结果显示,HtHCT基因在菊芋各组织中均有表达,其中茎中表达量最高.此外,HtHCT基因能够响应盐胁迫(150 mmol·L-1 NaCl)和干旱胁迫(20％ PEG-6000),NaCl处理24h的HtHCT基因表达量显著高于对照,PEG处理12h的HtHCT表达量与对照相比显著上调了10倍.

为探明燕山地区软枣猕猴桃天然种群表型性状的多样性,本研究对3个软枣猕猴桃天然种群内软枣猕猴桃资源的叶片、果实的14个质量性状和11个数量性状进行了观测及分析,结果表明:燕山地区三个软枣猕猴桃种群部分果实性状存在显著差异,而不同种群的叶片表型性状间未达到显著差异;11个数量性状变异系数在12.86％～30.40％,其中果心大小的变异幅度最大;14个质量性状变异系数在5.13％～77.80％,其中果实萼片宿存的变异幅度最大;11个数量性状多样性指数均值在1.69～3.10,其中叶柄长度的多样性最丰富;14个质量性状多样性指数均值在0.23～1.20,其中果实喙端形状的多样性最丰富.对燕山地区3个软枣猕猴桃种群的表型性状均值对比分析,结果表明花厂峪种群表型性状的变异系数最大,多样性指数最高,果实横径纵径较大,与其它种群相比具有较大的开发利用价值.本研究可为今后的软枣猕猴桃育种和群体遗传进化研究提供形态学方面的参考.

肉桂酰辅酶A还原酶(cinnamoyl-CoA reductase,CCR)是木质素合成代谢的关键酶.该研究以菊芋(Helianthus tuberosus L.)‘廊芋8号’为材料,克隆到1个菊芋的CCR基因,命名为HtCCR1(GenBank登录号为MN205540),其开放阅读框(ORF)长975 bp,编码324个氨基酸,其中含有FR_SDR_e保守结构域.系统进化分析表明,HtCCR1与向日葵CCR蛋白(XP_021989763.1)共聚于一支,二者亲缘关系最近.实时定量PCR分析表明,HtCCR1基因在菊芋茎和叶中的表达量显著高于在根和块茎中;盐(150 mmol· L-1 NaCl)胁迫处理6、12和24 h后,处理组HtCCR1基因的表达量均显著高于对照组;干旱(20％ PEG6000)胁迫6和12h后,处理组HtCCR1基因的表达较对照组均显著上调.成功构建pET-28a-HtCCR1原核表达载体,转化大肠杆菌BL21 (DE3)并诱导出了符合预期大小的蛋白,表明HtCCR1重组蛋白已成功表达.该研究结果为进一步研究HtCCR1基因的功能及利用基因工程手段调节菊芋中木质素的生物合成奠定了基础.

Walnut (Juglans regia L.) is an important woody oilseed species cultivated throughout the world. In this study, comparative transcript profiling was performed using high-throughput RNA sequencing technology at the following three stages of walnut fat synthesis in the “Lvling” walnut cultivar: the initial developmental stage (L1), the fast developing stage (L2), and the last developing stage (L3). A total of 68.18 GB of data were obtained on the three developmental stages, and 92% to 94% of clean data were able to be located to the reference genome. Further comparisons of the transcripts in the three libraries revealed that 724, 2027, and 4817 genes were differentially expressed between the L2 and L1 (L2vsL1), L3 and L2 (L3vsL2), and L3 and L1 (L3vsL1) samples, respectively. Through the GO gene enrichment analysis, differentially expressed genes (DEGs) in L2vsL1, L3vsL2, and L3vsL1 were enriched into 3, 0, and 2 functional categories, respectively. According to the KEGG enrichment analysis, DEGs in L2vsL1, L3vsL2, and L3vsL1 were annotated into 77, 110, and 3717 taxonomic metabolic pathways in the KEGG database, respectively. Next, we analyzed expression levels of genes related to fat synthesis. Our results indicated that ACCase, LACS, and FAD7 were the key genes related to fat synthesis. The high-throughput transcriptome sequencing of walnut in different developmental stages has greatly enriched the current genomic available resources. The comparison of DEGs under different developmental stages identified a wealth of candidate genes involved in fat synthesis, which will facilitate further genetic improvement and molecular studies of the walnut.

为探明影响核桃脂肪含量的主导脂肪酸因子,有效利用新疆核桃资源,采用索氏抽提法和气相色谱法测定26份不同基因型新疆核桃的脂肪含量及脂肪酸组成.结果表明:部分基因型新疆核桃间脂肪含量差异显著,以优21最高,为71.25 g/100 g,显著高于其它基因型.新疆核桃脂肪酸主要包括7种,以顺C18∶1n9e和顺C18∶2n6c为主,其中鸟什49的顺C18∶2n6c质量分数最高,为69.73％,纸皮的顺C18∶1n9c质量分数最高,为39.81％,显著高于其它基因型.脂肪酸顺C18∶1n9e的变异系数最大,为30.22％,脂肪酸C22∶0与顺C18∶1n9c闻的相关系数最大,脂肪酸C16∶1与脂肪含量相关性最大.通径分析结果表明:顺C18∶1n9c是影响脂肪含量的主导脂肪酸因子,脂肪与脂肪酸的线性回归方程为Y=71.721-2.169X2-172.882X4-74.5010X5+0.251X6-0.461X9.经验证该方程精度较高,可作为研究新疆核桃脂肪及脂肪酸因子关联性的模型.本研究探明了核桃脂肪和脂肪酸间的关联性,可为选育高油核桃品种提供科学依据.

In the reform of genetics experimental teaching,through setting up the experiment system of basic experiment-improved experiment—innovative experiment,designing the innovative experiment,developing the multimedia courseware,improving the teaching method of the experiment,assessing students' achievement comprehensively,arranging the teaching process rationally,strengthening laboratory construction,improving teacher's professional qualifications,the teaching effect was improved effectively and a very good achievement was obtained.

以早实核桃品种“绿岭”和“绿波”花粉为试材,采用离体培养方法,研究了培养基中不同比例蔗糖、硼酸、氯化钙对花粉萌发的影响,以明确核桃花粉萌发的适宜条件,为核桃花粉萌发找到适宜的培养基提供参考依据.结果表明:“绿岭”和“绿波”核桃花粉在蔗糖浓度为160 g·L-1时的发芽率最高,分别为48.09％和57.23％;“绿岭”和“绿波”花粉萌发最适宜的硼酸浓度分别为0.10、0.07 g·L-1;2个品种的花粉萌发最适宜的氯化钙浓度为0.20g·L-1.适宜“绿岭”核桃花粉萌发的最佳培养基为蔗糖160 g·L-1、硼酸0.10 g·L-1、氯化钙0.20g·L-1,在此培养基下“绿岭”核桃花粉萌发率达到61.06％;适宜“绿波”核桃花粉萌发的最佳培养基为蔗糖160 g·L-1、硼酸0.07g·L-1、氯化钙0.20g·L-1,在此培养基下“绿波”核桃花粉萌发率为72.71％.

为深入研究和开发利用新疆核桃资源,选育营养价值更高的核桃品种,采用索氏抽提法、气相色谱法、凯氏定氮法、HPLC法、酸水解法、消煮和ICP法,测定了6个新疆核桃优系以及新疆现行的优良品种温185的核仁脂肪与脂肪酸、蛋白质与氨基酸、淀粉与总糖、能量和矿质元素等主要营养物质,并进行了综合评价,结果表明:优系扎210、95和阿育王及对照温185核仁的脂肪含量在65.94 ～ 67.87 g/100 g,蛋白质含量在14.22 ～ 15.37 g/100 g,脂肪和蛋白质含量均达到国家核桃坚果质量特级标准;优系扎346和阿育王核仁脂肪中MUFA质量分数分别为25.89％、32.95％,显著高于温185 (20.46％),乌什49核仁脂肪中PUFA质量分数为78.69％,显著高于温185(71.69％);6个优系核仁蛋白质中EAA含量(3.00 ～4.56 g/100 g)均显著高于温185(2.14 g/100 g),是温185的1.4～2.1倍,除扎346外其他优系核仁蛋白质中TAA含量(12.10 ～ 16.92#100 g)均显著高于温185(10.84∥1O0 g),是温185的1.1 ～1.6倍;乌什49核仁中磷、钾、镁、铁、锰、铜和锌含量分别为444.71 mg/100 g,534.72 mg/100 g,197.18 mg/100 g,12.25 mg/100 g,3.44 mg/100 g,7.61mg/100 g,10.50 mg/100 g,均显著高于对照.采用PCA对6个新疆核桃优系及对照核仁主要营养物质进行综合评价,各优系营养价值由高到低依次为乌什49＞扎74＞ 95＞阿育王＞扎210＞扎346＞温185,6个新疆核桃优系核仁营养价值均优于对照温185.

In order to compare the chlorophyll fluorescence characteristics of different walnut rootstock varieties,and to select the vigorous rootstock varieties,the growth condition,photosynthetic characteristics,chlorophyll fluorescence and chlorophyll contents of tissue cultured seedlings of walnut rootstock varieties V and W were determined.The results showed that there was significant differences of plantlets growth between the 2 rootstock varieties.The seedling heights,ground diameter and the leaflets numbers of per compound leaf of V were extremely significantly less than that of W,which were 3.54 cm,4.86 mm,and 9 leaflets,respectively.However,the overall leaf area of V is larger than that of W.The content of chlorophyll b of V variety was extremely significantly less than that of W,which was 0.28mg/g.The daily mean values of Pn,Gs and Tr of V were also extremely significantly less than that of W,which were 8.78 μmol/(m2 · s),0.03 mol/(m2 · s),and 0.50 mmol /(m2 · s),respectively.The φPS Ⅱ and ETR of V were extremely significantly less than that of W,were 0.80 and 1.79,respectively.Through the comprehensive analysis,the stock variety W is superior to the variety V at the aspects of growth,photosynthesis and chlorophyll fluorescence characteristics.

[目的]研究春季低温胁迫下不同核桃品种叶片和子房超氧化物歧化酶(SOD)、过氧化物酶(POD)活性及丙二醛(MDA)、可溶性蛋白质含量的变化,为筛选抗霜冻能力强的核桃品种提供理论依据.[方法]以‘绿岭’、‘香玲’、‘辽宁1号’、‘上宋6号’、‘中林1号’和‘清香’6个核桃品种为试材,利用人工霜箱模拟春季霜冻害降温过程,设0、-1、-2、-3、-4、-5℃共6个低温处理,以18℃处理为对照,测定低温处理过程中叶片和子房SOD、POD活性及MDA、可溶性蛋白质的含量,采用隶属函数法对6个品种抗寒性进行综合分析.[结果]随温度的降低,6个供试核桃品种叶片和子房SOD、POD活性及可溶性蛋白质含量均呈现先升高后降低的趋势,但不同品种上升幅度与达到最高值的温度存在差异,而MDA含量呈现逐渐增高的趋势.‘绿岭’、‘香玲’、‘辽宁1号’、‘上宋6号’、‘中林1号’和‘清香’叶片SOD、POD活性及可溶性蛋白质含量达到最大值的温度分别为-4、-4、-3、-3、-3和-3℃,子房上述指标达到最大值的温度分别为-3、-3、-3、-3、-2和-2℃;叶片和子房MDA含量均在-5℃达到最大值;6个品种叶片平均隶属度分别为0.556,0.550,0.492,0.453,0.440和0.439,子房平均隶属度分别为0.509,0.482,0.473,0.445,0.423和0.374.[结论]供试6个核桃品种的抗霜冻能力为‘绿岭’＞‘香玲’＞‘辽宁1号’＞‘上宋6号’＞‘中林1号’＞‘清香’.

In order to breed transgenic plants with strong rooting ability and resistance to stress,we studied the anatomical structure of triploid poplar leaves.Three clones with one rolB and one rolCgene,six clones with two rolBand one rolCgene,and four clones with one rolB and two rolCgenes of transgenic triploid Populus tomentosatissue culture seedlings were used as materials.The results showed that the transgenic clones with different copies of rolBand rolCgenes did not perform certain regularities in direction and degree of variation.However,compared with triploid Populus tomentosa with one rolBand one rolCgene,the tightness of leaf palisade tissue structure of clone 101 Cincreased,and that of the clone 301 B,263B,363Band163 Cincreased significantly,while the other clones decreased significantly.This study can provide a reference for the selection of both rooting ability and excellent resistance of transgenic triploid Populus tomentosa.

为明确核桃园生草、覆盖效果,研究了核桃园行间种植苜蓿及苜蓿刈割后覆盖行内对土壤含水量、有机质、全N、全P、速效N、速效P等土壤理化性质的影响.结果表明:经过1年试验处理后,各处理0～20 cm土层土壤全磷、速效氮、20～40 cm土层的速效磷含量均存在极显著差异,0～20 cm和20～40 cm土层土壤全氮、0～20 cm土层土壤速效磷含量均存在显著差异.经过连续2年试验处理后,0～20 cm土壤有机质,3个土层土壤全磷、速效磷、速效氮和0～20 cm、20～40 cm土层土壤全氮含量均存在极显著差异.与对照相比,经过连续2年苜蓿覆盖后,苜蓿覆盖区不同土层的土壤含水量提高了2.22～4.08个百分比点,苜蓿覆盖区0～20 cm土层土壤有机质含量为14.67g/kg,提高了77.8％,土壤全磷含量为0.53 g/kg,提高了55.9％;苜蓿种植区20～40 cm土层土壤速效磷含量为17.17 mg/kg,提高了22.4％.表明核桃园种植苜蓿和用苜蓿覆盖能有效地提高土壤含水量和养分含量,增强土壤养分供给能力.

为充分利用新疆核桃种质资源,选育坚果综合品质优良的核桃品种,采用常规观测法、索氏抽提法、气相色谱法、凯氏定氮法和HPLC法,对8个新疆核桃优株坚果的16个外在品质指标和37个内在品质指标的变异情况及坚果综合品质进行了研究.结果表明:8个核桃优株坚果单果重、出仁率、坚果三径均值分别为14.73 g、51.67％和3.95 cm,脂肪、蛋白质含量均值分别为68.70％和13.66％,蛋白质中氨基酸总量(TAA)、人体必需氨基酸(EAA)含量均值分别为10.81 g/100g和3.11 g/100g,单不饱和脂肪酸(MUFA)和多不饱和脂肪酸(PUFA)含量均值分别为20.19％和71.25％,坚果外在品质中描述型性状和数值型性状变异系数分别为26.76％和14.34％,内在品质变异系数平均为19.84％.8个新疆核桃优株坚果品质变异比较丰富.主成分分析结果表明各优株坚果品质综合评价指数由高到低的顺序为:30号＞31号＞13号＞61号＞33号＞36号＞48号＞21号,可根据各优株不同的坚果品质特征开发出不同的核桃坚果产品.

为弄清不同土壤、立地类型对土壤含水率和苹果果实生长的影响,以‘长富2’苹果为试材,选取了粘壤土平地、粘壤土梯田、壤土平地、壤土梯田、沙土平地、沙土梯田、上层壤土下层尾矿砂平地7种不同土壤、立地类型苹果园,研究其对土壤含水率及苹果果实生长的影响.结果表明:粘壤土类型土壤含水率最高,壤土类型、上层壤土下层尾矿砂类型次之,沙土类型最低;上层壤土下层尾矿砂平地果实纵径、横径及体积均为最大,分别为6.858 cm、8.092 cm、301.82 cm3,粘壤土和壤土果实纵径、横径及体积次之,沙土果实纵径、横径及体积最小;粘壤土含水量与果实纵径、横径及体积多呈负相关关系,其中5月1日至5月15日粘壤土平地土壤含水率与果实纵径、横径及体积均呈极显著负相关,相关系数分别为-0.754、-0.827、-0.690;其他土壤、立地类型多呈正相关,其中5月16日至5月31日壤土梯田土壤含水率与果实纵径、横径及体积均呈显著或极显著正相关,相关系数分别为0.685、0.610、0.723;6月1日至6月28日上层壤土下层尾矿砂平地土壤含水率与果实纵径、横径及体积均呈显著或极显著正相关,相关系数分别为0.819、0.541、0.616.

为了给苹果园土壤水分管理提供依据,以‘长富2’苹果为试材,选取粘壤土平地、粘壤土梯田、壤土平地、壤土梯田、沙土平地、沙土梯田、上层壤土、下层尾矿砂平地7种不同立地、土壤类型苹果园,研究其对土壤含水量及苹果新梢生长的影响.结果表明:不同时期粘壤土平地的土壤含水量均极显著高于沙土平地、沙土梯田、上层壤土下层尾矿砂平地的土壤含水量,沙土平地、沙土梯田的土壤含水量均极显著低于其他立地、土壤类型.各立地、土壤类型长、中、短新梢均只有一次生长,迅速生长期基本一致,在4月初到5月底;除沙土平地和沙土梯田类型外,其他5种立地、土壤类型徒长新梢均出现了二次生长,第1次迅速生长期与其他新梢一致,第2次生长期在7月至8月.不同立地、土壤类型土壤含水量与苹果各类新梢长度均呈正相关,达到显著相关水平的时期多出现在新梢生长高峰期,其中,4月22日至5月13日,壤土梯田、沙土平地的土壤含水量与长梢长度的相关系数分别为0.968、0.966,5月14日至5月31日,壤土梯田的土壤含水量与徒长新梢长度的相关系数为0.986,壤土平地的土壤含水量与长梢长度的相关系数为0.980.