虉草品种营养器官解剖结构和种子休眠机理的比较研究
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摘要
虉草(Phalaris arundinacea L.)具有营养价值丰富、抗旱、抗寒、耐涝、耐盐碱等特性。可作为饲用、水土保持、移除河流污染、新能源燃料植物。在虉草营养器官解剖结构与其抗性和利用之间的关系基础上,确定了虉草具有较高的经济价值和生态价值潜力。但种子的休眠问题限制了对其的推广和应用。为了解决种子休眠,对通选7号和川草3号2个品种虉草种子外部形态、内部结构、种子生活力、种皮吸水性及胚性休眠进行鉴定的基础上,探索其休眠原因。用人工去除稃、磨破种皮、低温层积、超纯水浸种、KNO3浸种和赤霉素浸种等破眠方法进行了发芽检验。借助于蛋白质组学研究技术,深入分析了虉草种子休眠原因。本研究主要结果如下:
     1虉草根、茎维管束部分发达的通气组织是虉草耐涝性的主要解剖结构特征;茎、叶片角质化的表皮和叶表皮所含的丰富泡状细胞组是虉草具有抗旱性的主要显微特征。在生长后期根、茎所含丰富的木质素、纤维素是新能源燃料植物的主要特征。
     2通选7号种子褐色、川草3号种子黄色,种子形状都呈长椭圆形、表面附生微毛,顶端具突起,底部有蒂。种子具内外稃且膜质,外稃具芒,内、外稃在基部与种子紧密相连。从内部解剖结构看出,虉草种子具内外种皮,种皮与果皮紧密相连。种胚具有完整的胚根、胚轴、胚芽和盾片的结构,胚脂质,占种子总体积的1/3,其余都是淀粉质胚乳。
     3通选7号和川草3号种子的千粒重为、生活力、初始发芽率分别为0.607g、90.67%、9.30%和0.780g、92.00%、28.66%。前者的氧化还原酶类(SOD、POD和CAT)的含量低于后者。
     4通选7号种子的稃和种皮透水性差,存在机械性束缚,其休眠是由种皮障碍引起的综合性休眠。川草3号种子稃和种皮对种子萌发无显著影响,休眠是由胚部生理性休眠引起的综合休眠。
     5对2个品种种子进行物理和化学破眠方法的研究发现,通过去稃、磨破种皮、低温层积、流水冲洗、KNO3和GA3浸种都能不同程度地提高2个品种种子的发芽率。剥去稃后用砂纸磨破种皮是通选7号种子最佳的破眠方法,川草3号种子在剥去稃后再施加400mg/L的GA3综合处理后有最高的发芽率。
     6用400mg/L的GA3浸泡2个品种种子24h,都可显著促进种子的萌发。超纯水浸种24h对2个品种种子都无显著促进作用。
     7使用TCA/丙酮沉淀法提取蛋白,在pH4~7的IPG胶条上进行等点聚焦9h后可得到重复性好、分辨率高的蛋白质双向电泳图谱。硝酸银染色后经软件分析,可检测到900个以上蛋白点。
     8通过蛋白质双向图谱研究表明,所有处理和对照中,共检测到25个蛋白丰度表达差异在3倍以上的变化点。对其中的20个点进行了MALDI-TOF-TOF分析,16个得到了有效鉴定,通过功能分析,发现这些蛋白参与了能量与物质代谢、细胞骨架构成、信号传导、运输、防御和生物合成等诸多反应途径,相互联系构成了调控种子破除休眠庞大的蛋白质网络,促使种子萌发。
Phalaris arundinacea L. was with the characters of rich nutrition, drought, cold,waterlogging, saline-alkaline resistance.They could be used as forage,conservation ofwater and soil,river pollution removing and new energy plants.On the basis of therelationship between anatomical structure of vegetative organ and stress resistence,usevalue of Phalaris arundinacea L. determined its high potentional economical andecological value,but the seed dormancy restricted its popularization and application.Seed dormancy reason of two varieties was identified on the basis of the external andinternal structure of seed, seed vigor, seed coat absorbent and embryo dormancy.Thegermination test was carried out by removing the lemma artificially, abrading seed coat,low temperature lamination and soaking in ultrapure water, KNO3and GA3. By usingproteomics research technology, analysed the seed doemancy mechanism intensively.Themain results were as follows:
     1. Good ventilation organization visible in root cortex cell gaps and air cavity, primaryxylem epigenetic catheter and medullary cavity were the main microscopic characteristicsof waterlogging resistance. The hornificated epidermis of stem and leaves and its abundantbulliform cell groups were the main anatomical structure reasons for drought resistance. Alarge numbers of wood fibers in vascular bundles of stem and leaves were majormicroscopic characteristics of new energy plants.
     2. The Phalaris arundinacea L. was with projection apex and pedicle bottom、brownor yellow coloured, oblong shaped and smoothly surfaced. The seeds with membranousglumes, lemma and the lemma was awned and they closely linked at the base of caryopsis.Seeing from the anatomical structure, Phalaris arundinacea seeds were composed of innerand outer testa, which were closely connected with pericarp. The embryo had integritystructure of radicle, hypocotyls, germ and scutellum and it accounted for1/3of totalvolume of the seed. The rest of the seed was starchy endosperm.
     3. The thousand seed weight, seed viability and initial seed germination ofP.arundinacea L. cv. Tongxuan No.7and P.arundinacea L. cv. Chuancao No.3seed were0.607g,90.67%,9.30%and0.780g,92.00%,28.66%separately.The former’sOxidoreductases (SOD, POD and CAT) content was lower than that of the latter’s.
     4. The water permeability of lemma and seed coat was poor and they had mechanical restraint on P.arundinacea L. cv. Tongxuan No.7seed, so its dormancy was caused by thebarrier of seed coat comprehensivly. P.arundinacea L. cv. Chuancao No.3seed’s lemmaand coat had no significant effect on seed germination, the dormancy was caused by thephysiological embryo dormancy comprehensivly.
     5. The physical and chemical dormancy breaking methods were carried out on twovarieties of seeds, the results showed that removing the lemma、worning the seed coat, coldstratification, water flushing, KNO3and GA3pretreatment could significantly enhanced thegermination rate of two varieties. Removed the lemma, and then abraded the seed coat wasseen as the best dormancy breaking method of P. arundinacea L.cv. Tongxuan No.7seed.P.arundinacea L.cv. Chuancao No.3seed had the highest germination rate under removingthe lemma and then soaking in400mg/L GA3.
     6. Soaking in400mg/L GA324h could significantly promote the seed germination oftwo varieties. Soaking in ultrapure water24h had no significant effect on two varieties ofseeds.
     7. Using TCA/acetone precipitation method extracted the protein, the pH4~7IPG stripfor isoelectric focusing9h could obtain good repeatable and high resolutionaltwo-dimensional electrophoresis images. After staining with silver nitrate and the softwareanalysis, colud detect more than900protein spots.
     8. By studying in all the treatment and control’s two-dimensional electrophoresisimages, found that there were25protein spots expressed differentially more than3times inabundance changes. Analysing the20spots by MALDI-TOF-TOF,16got the effectiveidentification and through the functional analysis, found that these proteins were involvedin energy metabolism, cytoskeleton, signal transduction, transportation, defense andbiosynthesis pathways, they mutually associated and constituted the enormous seeddormancy breaking regulatory network and promoted the seed germination.
引文
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