青扦花粉萌发及花粉管生长过程中的钾离子流及BaCl_2和TEA的影响

本文以裸子植物青扦花粉为材料,运用非损伤、选择性电极技术探究了花粉水合、萌发及花粉管生长过程中的钾离子流,以及抑制剂处理后的影响。研究结果显示:(1)钾离子通道抑制剂BaCl_2和TEA(氯化四乙胺)处理推迟花粉萌发;(2)BaCl_2和TEA处理后,青扦花粉萌发和花粉管伸长均受到抑制;(3)选择性电极检测结果显示对照花粉不同发育时期钾离子均内流,即花粉水合、萌发及花粉管生长均需大量的钾离子。BaCl_2和TEA处理后,花粉的钾离子流动态也随之发生改变。培养3~9 h,钾离子呈明显的外流,12~21 h钾离子内、外流基本平衡,24 h钾离子内流。抑制剂处理导致的钾离子流的紊乱使得花粉不能正常萌发、花粉管不能正常生长。因而,钾离子在青扦花粉萌发及花粉管生长过程中具有重要的调控作用。

钙离子对青杄花粉萌发和花粉管生长的影响

本文以成熟的青杄花粉为试验材料,用不同浓度钙拮抗剂EGTA和抑制剂AlCl_3进行处理,观察花粉的萌发和花粉管的生长情况,同时用选择性电极测定了不同发育时间内花粉管顶端钙离子流,以及EGTA和AlCl_3对钙离子流的影响,以探讨钙对青杄花粉生长发育的调控机制。结果发现:(1) 0.5 mmol·L^(-1) EGTA处理花粉后,会促进花粉萌发,萌发率升高;但花粉管长度低于对照组;1 mmol·L^(-1) EGTA处理后,花粉萌发率和花粉管长度都低于对照组;0.1 mmol·L^(-1)和0.5 mmol·L^(-1) AlCl_3处理后,花粉萌发率和花粉管长度都会显著降低;(2)通过非损伤微测技术发现,对照花粉在水合时期(培养3,6,9 h)钙离子外流,在萌发及快速伸长期钙离子内流(培养12,15,18 h);EGTA和AlCl_3培养花粉后,花粉的水合期、萌发期和花粉管的伸长期的钙流状况会出现异常,其中包括钙流的内流和外流的状况以及流速,从而导致各个时期花粉的萌发率及和花粉管长度的改变,导致花粉管的生长异常。(3)对照花粉管顶端出现Ca^(2+)浓度梯度,而0.5 mmol·L^(-1) EGTA处理的花粉会相比对照组提前在花粉管顶端出现Ca^(2+)浓度梯度,以促进花粉萌发;1 mmol·L^(-1 )EGTA,0.1 mmol·L^(-1) AlCl_3和0.5 mmol·L^(-1) AlCl_3处理后Ca^(2+)浓度梯度消失。由此表明花粉的不同生长时期对钙离子的需求不同,且钙离子对花粉管的萌发及生长起着至关重要的作用。

EGTA对‘秦冠’苹果花粉管壁主要成分的影响

本文以不同浓度Ca^2+螯合剂EGTA处理‘秦冠’苹果成熟花粉,观察其对花粉管细胞壁主要成分的影响。在低浓度EGTA(400μmol·L^-1EGTA)处理下纤维素、胼胝质、酯化果胶无明显变化,酸性果胶、阿拉伯半乳糖蛋白在花粉管壁两侧的荧光强度增强。在高浓度EGTA(2000μmol·L^-1 EGTA)处理下苹果花粉管上的纤维素无明显变化;胼胝质在花粉管顶端富集;花粉管顶端及两侧壁酸性果胶、酯化果胶浓度增加;花粉管壁两侧及基部的阿拉伯半乳糖蛋白荧光强度增加。进一步用傅立叶红外光谱技术分析验证了花粉管顶端细胞壁化学成分的变化。因此作者推测EGTA通过干扰钙的含量进而调控细胞壁多糖成分,进而造成细胞壁的物理机械强度以及延展性的变化,最终达到调节花粉萌发及花粉管生长的作用。

Fingerprinting 146 Chinese chestnut(Castanea mollissima Blume)accessions and selecting a core collection using SSR markers

Chinese chestnut is an important nut tree around the world.Although the types of Chinese chestnut resources are abundant,resource utilization and protection of chestnut accessions are still very limited.Here,we fingerprinted and determined the genetic relationships and core collections of Chinese chestnuts using 18 fluorescently labeled SSR markers generated from 146 chestnut accessions.Our analyses showed that these markers from the tested accessions are highly polymorphic,with an average allele number(N_(a))and polymorphic information content(PIC)of 8.100 and 0.622 per locus,respectively.Using these strongly distinguishing markers,we successfully constructed unique fingerprints for 146 chestnut accessions and selected seven of the SSR markers as core markers to rapidly distinguish different accessions.Our exploration of the genetic relationships among the five cultivar groups indicated that Chinese chestnut accessions are divided into three regional type groups:group I(North China(NC)and Northwest China(NWC)cultivar groups),group II(middle and lower reaches of the Yangtze River(MLY)cultivar group)and group III(Southeast China(SEC)and Southwest China(SWC)cultivar groups).Finally,we selected 45 core collection members which represent the most genetic diversity of Chinese chestnut accessions.This study provides valuable information for identifying chestnut accessions and understanding the phylogenetic relationships among cultivar groups,which can serve as the basis for efficient breeding in the future.

The MADS-box transcription factor CmAGL11 modulates somatic embryogenesis in Chinese chestnut(Castanea mollissima Blume)

Somatic embryogenesis(SE)is an effective approach of in vitro regeneration that depends on plant cell totipotency.However,largely unknown of molecular mechanisms of SE in woody plants such as Chinese chestnut(Castanea mollissima Blume),limits the development of the woody plant industry.Here,we report the MADS-box transcription factor Cm AGL11 in Chinese chestnut.Cm AGL11 transcripts specifically accumulated in the globular embryo.Overexpression of Cm AGL11 in chestnut callus enhanced its SE capacity,and the development of somatic embryos occurred significantly faster than in the control.RNA-seq results showed that Cm AGL11 affects the expression of several genes related to the gibberellin,auxin,and ethylene pathways.Moreover,the analysis of DNA methylation status indicated that the promoter methylation plays a role in regulation of Cm AGL11 expression during SE.Our results demonstrated that Cm AGL11 plays an important role in the SE process in Chinese chestnut,possibly by regulating gibberellin,auxin,and ethylene pathways.It will help establish an efficient platform to accelerate genetic improvement and germplasm innovation in Chinese chestnut.

板栗实时定量PCR内参基因的筛选与验证

实时荧光定量PCR技术具有高灵敏性和高特异性,被广泛应用于基因表达分析,选取合适的内参基因是利用该技术准确分析目的基因表达变化的前提。本文以板栗的7种不同组织器官(根、茎、叶、种子、花芽、雌花和雄花)为材料,采用qRT-PCR技术分析了板栗中6个候选内参基因(EF1α、TUA、TUB、UBQ、18S rRNA和Actin)的表达趋势,运用geNorm、NormFinder和BestKeeper 3种软件对候选内参基因的表达稳定性进行了综合分析,并利用目的基因CmSPL9对筛选出的内参基因进行验证。结果表明:Actin和EF1α在板栗不同组织器官中稳定性最佳, Actin、EF1α或Actin+EF1α可作为板栗的内参基因进行后续试验研究。