甘蔗热带种金属硫蛋白家族基因的克隆及响应重金属胁迫的表达分析

金属硫蛋白是一类富含巯基的低分子量蛋白,在植物的重金属解毒及细胞氧化还原调控等方面起重要的作用。本研究以甘蔗热带种Badila组培苗为材料,分别测定了其在CdCl2、ZnSO4和CuCl2水溶液培养条件下地上部和地下部的重金属含量,结果显示其对上述3种重金属有较强的耐受与富集能力。继而克隆了ScMT1(登录号为KJ504373)、ScMT2-1-5(登录号为MH191346)和ScMT3(登录号为KJ5043704)3个金属硫蛋白家族基因,它们分别属于植物MT亚家族中的MT1、MT2和MT3型基因。ScMT1含有1个内含子和2个外显子,开放阅读框(Open Reading Frame,ORF)长228 bp,编码75个氨基酸;ScMT2-1-5含有2个内含子和3个外显子,ORF长246 bp,编码81个氨基酸;ScMT3含有1个内含子和2个外显子,ORF长198 bp,编码65个氨基酸。RT-qPCR显示,Cd2+胁迫下,在甘蔗地上部和地下部,ScMT2-1-5均连续显著上调表达,而ScMT1的上调应答出现延迟。ScMT3在地上部的上调应答出现延迟,在地下部呈“扬-抑”趋势,提示甘蔗响应Cd^2+胁迫过程中ScMT2-1-5起更积极的作用,ScMT1参与胁迫后期的分子响应,而ScMT3不起主导作用。Cu^2+胁迫下,地上部ScMT1连续显著上调表达,ScMT2-1-5和ScMT3呈总体上调的表达趋势;地下部,ScMT1和ScMT2-1-5的上调表答均出现延迟,仅在胁迫后期显著上调表达,而ScMT3仅在胁迫前期显著上调表达。该结果提示了ScMT1、ScMT2-1-5和ScMT3在Cu2+胁迫响应过程中的协作关系,三者共同参与了地上部的胁迫响应,其中ScMT1起更积极的作用;此外三者还先后参与了地下部对Cu^2+胁迫的分子响应。Zn^2+胁迫下,ScMT1和ScMT3分别仅在地上部和地下部显著上调表达;ScMT2-1-5在地上部和地下部均呈“扬-抑”的应答趋势;提示了在甘蔗响应Cd^2+胁迫应答过程中ScMT1和ScMT3分别在地上部和地下部起主要作用,ScMT2-1-5参与了胁迫前期的分子响应。ScMT1、ScMT2-1-5和ScMT3在甘蔗不同组织中及在重金属(Cd^2+、Zn^2+或Cu^2+)不同累积水平下呈现出相似或互补的应答特性,提示上述甘蔗MT家族不同成员在重金属解毒及细胞氧化还原调控等方面产生了功能分化,且三者在应对过量Cd^2+、Zn^2+或Cu^2+对甘蔗组织造成伤害的过程中存在时空上的协同作用。该研究为深入理解多倍体植物甘蔗中MT家族各成员基因在重金属耐受过程中的协同作用机制奠定了基础。     

Exogenous application of allelopathic water extracts helps improving tolerance against terminal heat and drought stresses in bread wheat (Triticum aestivum L. Em. Thell.)

The allelopathic water extracts (AWEs) may help improve the tolerance of crop plants against abiotic stresses owing to the presence of the secondary metabolites (i.e., allelochemicals). We conducted four independent experiments to evaluate the influence of exogenous application of AWEs (applied through seed priming or foliage spray) in improving the terminal heat and drought tolerance in bread wheat. In all the experiments, two wheat cultivars, viz. Mairaj‐2008 (drought and heat tolerant) and Faisalabad‐2008 (drought and heat sensitive), were raised in pots. Both wheat cultivars were raised under ambient conditions in the wire house till leaf boot stage (booting) by maintaining the pots at 75% water‐holding capacity (WHC). Then, managed drought and heat stresses were imposed by maintaining the pots at 35% WHC, or shifting the pots inside the glass canopies (at 75% WHC), at booting, anthesis and the grain filling stages. Drought stress reduced the grain yield of wheat by 39%–49%. Foliar application of AWEs improved the grain yield of wheat by 26%–31%, while seed priming with AWEs improved the grain yield by 18%–26%, respectively, than drought stress. Terminal heat stress reduced the grain yield of wheat by 38%. Seed priming with AWEs improved the grain yield by 21%–27%; while foliar application of AWEs improved the grain yield by 25%–29% than the heat stress treatment. In conclusion, the exogenous application of AWEs improved the stay green, accumulation of proline, soluble phenolics and glycine betaine, which helped to stabilize the biological membranes and improved the tolerance against terminal drought and heat stresses.     

Identification and quantification of anthocyanins in different coloured cultivars of ornamental kale (Brassica oleracea L. var. acephala DC)

Anthocyanins are responsible for the colour of many fruits, vegetables, flowers, and coloured-leaved trees. Ornamental kale (Brassica oleracea L. var. acephala DC) is widely cultivated for its colourful inner leaves. To investigate the relationship between the degree of colouration and anthocyanin distribution, content, and composition in ornamental kale, the authors studied the pigment characteristics of five cultivars with different coloured leaves (white, pink, red, purple, and purple-black). Microscopy observation, spectrophotometer, and high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of fresh inner leaves revealed that pink, red, and purple colourations were associated with high levels of anthocyanin, while purple-black was the result of the combination of anthocyanins and chlorophyll. In the coloured cultivars, anthocyanins were abundant mainly in the first and second cell layers below the epidermis in both the hypocotyls and inner leaves. No anthocyanin was found in the white-leaved phenotype cultivar. Anthocyanin content increased as leaf colour deepened from pink, red, to purple cultivars, which had little chlorophyll and carotenoid. The authors identified eight anthocyanins in the four coloured cultivars, including one non-acylated, four monoacylated, and three diacylated cyanidin glycosides. Cyanidin-3-(sinapoyl)(feruloyl)-diglucoside-5-glucoside was the most abundant anthocyanin in the four coloured cultivars followed by cyanidin-3-(sinapoyl)-diglucoside-5-glucoside. The analysis of anthocyanin accumulation characterisation provides important information on evaluating colouration patterns in coloured plants, and will be helpful for breeding desired leaf colours in ornamental kale.     

白屈菜CmFAD2基因的克隆及植物转化载体构建

通过对白屈菜低温应答过程的转录组分析发现膜脂不饱和化相关基因的表达在一定过程中发生变化,脂肪酸去饱和酶基因FAD2在随温度的变化趋势为正"V"型,且表达量变化显著。利用NCBI等在线软件对序列进行相关生物学信息分析,并对白屈菜FAD家族成员FAD2基因的完整开放阅读框(ORF)进行克隆,并命名为CmFAD2。选用克隆载体pMD-19-T,转化大肠杆菌DH5α,测序验证序列正确性及完整性。将目的基因与植物表达载体pRI-201-AN连接构建重组DNA pRI-201-AN-Cm FAD2,电击法转化农杆菌LBA4404,利用菌液PCR法验证成功。该基因可作为药用植物抗寒品种创制的候选基因。     

大花君子兰叶绿体基因组及其特征

采用Illumina MiSeq测序平台对大花君子兰（Clivia miniata）叶片总DNA进行测序，通过组装获得了其叶绿体基因组（cpDNA）全长序列（158 114 bp）。对其cpDNA注释得到135个基因，包含87个蛋白编码基因、40个tRNA基因和8个rRNA基因。采用生物信息学方法对获得的cpDNA进行简单序列重复（SSR）分析和密码子偏好性分析。结果显示：①大花君子兰cpDNA中共有61个SSR位点，其中单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复数分别为38、9、2、8、3和1个，多数SSR分布在基因间隔区；②大花君子兰cpDNA密码子偏爱以A或U（T）结尾，亮氨酸使用频率最高，半胱氨酸使用频率最低。基于24种植物的cpDNA全长和23种植物的叶绿体ycf2基因序列进行系统发育分析，结果显示大花君子兰与石蒜科植物在同一分支，显示最近的亲缘关系，支持大花君子兰属于石蒜科。基于叶绿体ycf2的系统发育分析结果与基于cpDNA全长的系统发育分析研究结果大部分相同，支持ycf2基因可以代替cpDNA全长用于植物系统发育分析。     

芸薹属及其他异源多倍体植物的基因表达特征研究进展

远缘杂交及异源多倍化导致许多重要作物的起源与进化，而芸薹属栽培异源四倍种是研究作物异源多倍化的模式系统之一。异源多倍体是如何调控及协调来自不同二倍体祖先的不同基因组的遗传行为及基因表达，是过去二十年间的研究热点和重要的生物学问题。利用不断发展的分子生物学技术，一方面揭示出芸薹属及其他多倍体物种基因组表现出动态性质，即在形成初期及长期进化过程中持续发生遗传及表观遗传的变化；另一方面发现异源多倍化过程中伴随着大量的基因表达模式改变，包括非加性表达、超亲表达、表达水平显性、部分同源偏向表达、基因剂量平衡效应等现象。上述基因组结构、表观遗传改变以及基因表达模式的调控，使新产生的多倍体得以成功进化为新物种。     

正交设计优化青蒿SRAP-PCR反应体系及引物筛选

为了建立青蒿的SRAP最佳扩增体系,并筛选出SRAP多态性引物,本研究以青蒿叶片DNA为模板,采用正交试验设计,以Mg^2+、dNTP Mix、Taq DNA聚合酶、引物和DNA模板5种因素5个水平,对青蒿SRAP反应体系进行研究。结果表明,青蒿SRAP-PCR最佳反应体系为:引物0.6μmol/L、Mg^2+2.0 mmol/L、模板DNA 5.1 ng、Taq DNA聚合酶2.0 U、dNTPs 0.25 mmol/L,总体积为25μL。各因素对扩增反应均有不同影响,其中引物浓度的影响最大,dNTPs的影响最小。运用该体系对不同种质资源的青蒿进行验证,证明该体系稳定可靠,并在30个引物组合中筛选出了25对扩增条带清晰,多态性丰富的引物组合。这一结论为今后利用SRAP标记技术进行青蒿分子遗传学研究提供了科学依据。     

槟榔间作条件下不同修剪方式对可可生长和产量的影响

以槟榔间作可可条件下的可可为研究对象,比较不同修剪方式对其生长、光合作用及产量的影响。结果表明:中度修剪提高可可株高、干周和主枝粗度,重度修剪和仅控冠幅降低可可干周和主枝粗度。中等修剪可可叶片Pn显著高于轻度修剪和仅控冠幅,可能由于修剪提高了新梢叶片叶肉细胞光合作用能力。中度修剪可可徒长枝重量分别比轻度修剪、重度修剪和仅控冠幅显著降低了69.27%、54.69%和57.03%,大大降低了养分消耗,提高产量的同时节省了修剪工作量。综上所述,在槟榔间作可可条件下,采用中度修剪有利于提高光合作用效率,形成健康平衡树形,降低徒长枝养分消耗,提高产量,为槟榔间作可可节本增效技术研究与优化提供依据。     

中药对肉仔鸡脂质代谢及肉品质的影响

将120只1周龄肉仔鸡随机分成4组,每组30只,前3组(试验Ⅰ、Ⅱ、Ⅲ组)分别喂以含1%的3种不同中药组方的饲粮,第Ⅳ组为对照组,只喂基础日粮.试验期6周.结果(1)与对照组相比,Ⅰ组血清TC、LDL-C极显著下降(P＜0.01);Ⅱ组血清TC、LDL-C极显著下降(P＜0.01),TG也下降(P＜0.05),HDL-C显著升高(P＜0.01);Ⅲ组血清LDL-C极显著下降(P＜0.01),HDL-C则显著升高(P＜0.01);试验Ⅰ～Ⅲ组血清总脂都极显著下降(P＜0.01);与Ⅱ组相比,Ⅰ组血清LDL-C、Ⅲ组TC存在显著差异(P＜0.01).(2)与对照组相比,胸肌组织中,各试验组TG极显著下降(P＜0.01),Ⅱ组TC显著下降(P＜0.05);腿肌中,各组TC显著下降(P＜0.05),TG下降极显著(P＜0.01);肝脏中各组TC、TG均极显著下降(P＜0.01);与Ⅱ组相比,Ⅰ组肝TG差异极显著(P＜0.01);Ⅲ组胸肌TG、肝TG有显著差异性(P＜0.05).(3)7周龄末鸡体重与对照组相比,Ⅱ组差异极显著(P＜0.01),Ⅲ组差异显著(P＜0.05);Ⅱ组料肉比比对照组极显著下降(P＜0.01).(4)肌肉中的营养成分,Ⅰ、Ⅱ组腿肌中粗脂肪比对照组极显著下降(P＜0.01),Ⅱ组腿肌中粗蛋白含量显著升高(P＜0.01).结论中药组方能不同程度地调整鸡脂质代谢,提高生产性能和改善鸡肉品质.在3个不同中药组方中,综合效果Ⅱ方最优.