结缕草生物技术研究进展

结缕草属植物具有耐干旱、耐盐和耐践踏等优良特性。最近的研究表明,基因转移与重组技术在结缕草遗传改良中具有巨大应用潜力;结缕草遗传连锁图谱构建取得重要进展;基因克隆和基因资源研究正在展开。结合当前有关结缕草生物技术研究现状,综述了结缕草基因工程和基因资源方面的研究进展,讨论了存在的问题并展望其前景。     

大穗和中华结缕草的群落特征及种内分异研究

大穗结缕草为盐生植物,主要生长在沿海潮沟两侧的滨海盐土地带上,其区系组成绝大多数种类属于盐生植物,为海滩原生植物群落。中华结缕草为耐盐的优良草坪植物,其野生群落主要分布在沿海2~40m的滨海阶地上。其生态幅较宽,土壤pH在7.2~9.2之间。中华结缕草群落为单优群落,盖度多在60%~90%。中华结缕草种内存在十分丰富的变异,采用模糊聚类的方法将17份草样划分为6类,各类间差异很大。主分量分析表明有性繁殖器官如果穗高度和营养器官特征如叶长和匍匐茎扩展速度是研究中华结缕草种内分异的重要指标。这2种植物可作为沿海城市建植草坪的优良植物。     

Relative costs and benefits of clonal integration of Zoysia japonica under various N:P ratios

Clonal plants adaptively respond to abiotic stress, but to date little is known about under what circumstanses clonal integration is beneficial, or costly. To study the costs and benefits of clonal integration on clonal growth, we cultivated Zoysia japonica under three ratios of N:P (N:P ≈ 7, 14:1 and 21:1), and four types of stolon severing treatments (connected stolon CS, light severing LS, moderate severing MS, serious severing SS). The results showed that Z. japonica performed best at a low ratio of N:P (N:P ≈ 7:1). When the stolons were connected (CS), the growth of primary A‐ramets, multiple‐nodes and stolons benefited from clonal integration; however, the growth of primary B‐ramets on the primary stolons, A and B ramets was significantly reduced (p < 0.05). In the moderate stolon severing treatment MS_7:1 (N:P ≈ 7:1), clonal integration appeared more cost effective than in all other treatments. On the whole, with increasing stolon severing intensity, the cost of clonal integration increased and the clonal growth of Z. japonica declined significantly. The pattern of biomass allocation may be useful for Z. japonica to adapt to the various environments, and clonal integration plays a significant role under adverse environmental conditions.     

Carbon stock and seasonal dynamics of carbon flux in warm-temperature tussock ecosystem in Shandong Province,China

了解山东省草地生态系统碳库现状和碳通量变化规律对于全国尺度草地生态系统碳源/汇核算有着重要的意义。该研究采用野外面上调查取样和固定加强点静态箱法(LI-840红外分析仪联用)相结合的方法, 分析了山东省暖性草丛生态系统的固碳现状、碳通量季节动态以及净生态系统CO₂交换(NEE)对各种环境因子的响应。研究结果表明: 山东暖性草丛生态系统平均碳密度为2.74 Mg C·hm ^-2, 碳密度的构成排序为土壤碳密度(89%) >生物量碳密度(9%) >凋落物碳密度(2%), 山东暖性草丛碳库总储量约为15.88 Tg C; 结缕草(Zoysia japonica)暖性草丛生态系统NEE的季节动态总体表现为夏季低, 冬季高, 非生长季节(11月至次年4月)向外界净排放CO₂, 表现为碳源效应; 生长季节(4-9月)则为净吸收CO₂ , 表现为碳汇效应, 峰值月份的平均固碳速率在-2.58- -4.46 μmol CO₂·m ^-2·s ^-1之间; 2012和2013年泰山小流域暖性草丛NEE年平均值分别为-0.43 μmol CO₂·m ^-2·s ^-1和-0.31 μmol CO₂·m ^-2·s ^-1, 都表现为碳汇效应; 光合有效辐射(PAR)、大气温度(T_a)、饱和水汽压差(VPD)和土壤10 cm深度温度(T_s)和含水量(W)是结缕草暖性草丛生态系统NEE动态的主要影响因素, 但不同月份NEE动态的影响因素各异, 且因子间存在着互作效应, 主成分分析表明, NEE的季节动态主要受温度、水分和光强等因子控制。     

假俭草和结缕草营养器官结构对抗逆性的影响

利用光学显微镜和电镜技术,采用石蜡切片法,叶片离析法等实验技术对假俭草和结缕草的茎,叶等营养器官的解剖结构特征和表皮微形态特征作了研究;通过角质层厚度,维管束数目,气孔指数,泡状细胞等数据分析它们之间的抗逆性差别;首次从微形态角度初步了解假俭草和结缕草及其不同种群之间抗旱力,耐践踏性差异的原因。     

Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation

Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass that spreads by stolons and rhizomes. By exploring the potential of direct shoot formation from stolons, we developed a straightforward and efficient transformation protocol without callus induction and propagation. Sterilized stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Both green and albino shoots were directly regenerated from the infected stolon nodes 4–5 weeks after hygromycin selection. Greenhouse-grown plants were obtained 10–12 weeks after Agrobacterium-mediated transformation. Based on the number of transgenic plants obtained and the number of stolon nodes infected, a transformation frequency of 6.8% was achieved. Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern blot hybridization analyses. Expression of the transgenes was confirmed by RT-PCR analysis and GUS staining. The new transformation system opens up new opportunities for the functional characterization of genes and promoters and the development of novel germplasm in zoysiagrass.     

黄河三角洲大穗结缕草解剖结构的研究

目前对于草坪草的抗性研究多数集中在其生理方面的变化,而结合各抗性品种营养器官结构进行比较研究却报道较少。从采集自山东省无棣县沿海滩涂的大穗结缕草的营养器官解剖结构和微形态特征的研究中分析它的耐盐、耐寒、耐旱、耐践踏和弹性好的原因,从形态解剖的角度来认识大穗结缕草的优良特性。     

日本结缕草总RNA提取和mRNA分离方法的优化

介绍了分别以日本结缕草根部、叶部、匍匐茎等为材料的总RNA提取的改良步骤,质量和浓度检测及其注意事项,并采用Promega公司的Poly ATract Systems Ⅲ(Z5300)试剂盒进行mRNA分离,尽管这一方法成熟可行,但仍存在洗脱体积较大,常需先沉淀浓缩后才能进行后续试验(如cDNA合成),在制备少量mRNA时回收率较低等问题。据此,对mRNA分离方法作了改良,介绍了mRNA分离的优化步骤,通过增加体系形成了一种更适合于日本结缕草mRNA分离的方法,从而为日本结缕草的分子生物学研究提供基础资料。结果证明所提取的总RNA和mRNA完整,质量较高,并应用到日本结缕草cD-NA文库的构建。     

Isolation of cDNA and enzymatic properties of betaine aldehyde dehydrogenase from Zoysia tenuifolia

We isolated cDNAs encoding betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from the salt-tolerant Poaceae, Zoysia tenuifolia by polymerase chain reactions. Zoysia betaine aldehyde dehydrogenase 1 (ZBD1) is 1892bp long and codes for 507 amino acids. The deduced amino acid sequence of ZBD1 is 88% similar to the sequence of rice BADH. Ten cDNA clones were isolated from a cDNA Library of salt-treated Z. tenuifolia by using the ZBD1 fragment as a probe. The proteins coded in some clones were more homologous to BBD2, the cytosolic BADH of barley, than to ZBD1. To investigate their enzymatic properties, ZBD1 and spinach BADH were expressed in Escherichia coli and purified. The optimal pH of ZBD1 was 9.5, which was more alkaline than that of spinach BADH. ZBD1 was less tolerant to NaCl than spinach BADH. ZBD1 showed not only BADH activity but also aminoaldehyde dehydrogenase activity. The Km values of ZBD1 for betaine aldehyde, 4-aminobutyraldehyde (AB-ald), and 3-aminopropionaldehyde (AP-ald) were 291, 49, and 4.0 microM, respectively. ZBD1 showed higher specific activities for AB-ald and AP-ald than did spinach BADH.     

立枯丝核菌(Rhizoctonia solani)对日本结缕草(Zoysia japonica steud.)的侵染过程研究

【目的】了解立枯丝核菌(Rhizoctonia solani) AG1 IA对日本结缕草(Zoysia japonica steud.)的侵染过程及其引起的病害症状,为进一步从分子水平研究该菌的病理学侵染机制和草坪草抗病分子育种提供理论基础。【方法】通过根部接种法促使立枯丝核菌与日本结缕草无菌苗建立侵染关系,从而对其病叶率、病株率及病情指数进行统计分析,同时结合组织染色透明技术及植物组织石蜡切片对立枯丝核菌的侵染过程、感染方式进行研究。【结果】立枯丝核菌AG1 IA的侵染过程主要为：菌丝吸附在植物组织表面,并沿组织表面定向生长,形成侵染结构——侵染垫与组织建立密切的侵染关系。菌丝通过细胞间隙侵入植物组织内部,主要侵染植物皮层细胞及除木质部导管以外的整个维管束系统。结缕草的地上部分与地下部分组织对立枯丝核菌的侵染显现不同的寄主反应。【结论】立枯丝核菌的侵染过程主要包括吸附、定向生长、渗透、定殖4个部分;立枯丝核菌的侵染主要引起结缕草叶片病症;结缕草病变与菌丝直接侵染无直接联系,表明该菌具有复杂的侵染机制。