Less and shrunken pollen 1(LSP1) encodes a member of the ABC transporter family required for pollen wall development in rice(Oryza sativa L.)

Pollen fertility is an agronomic trait that strongly influences rice yield. Recent studies have revealed that the development of the pollen wall is required for pollen fertility and is regulated by several genes. However, the mechanisms underlying pollen and pollen wall development in rice remain largely unknown. In the present study, a point mutation in a gene on chromosome 1 was identified that resulted in the production of less and shrunken pollen(LSP) and led to defects in pollen wall formation. This gene was named LSP1 and was found to encode a member of the adenosine triphosphate-binding cassette(ABC)transporter G subfamily, OsABCG3. Two other loss-of-function mutants of LSP1/OsABCG3,generated using CRISPR/Cas9 technology, showed the same male sterile phenotype. The LSP1/OsABCG3 gene showed a spatio-temporal expression pattern in the developing anthers, and is an ortholog of the Arabidopsis genes At ABCG1 and At ABCG16, which play an important role in pollen wall development. Mutation of LSP1/OsABCG3 affected the expression of several genes involved in pollen and pollen wall formation. These results suggest that LSP1/OsABCG3 is critical for normal pollen fertility and shed light on the molecular mechanisms underlying rice pollen wall development.

拟南芥雄性不育突变体opw(only pollen wall)候选突变基因的克隆

在T-DNA插入突变体Salk_059463株系的群体中,筛选到两株雄性不育突变体,对TDNA序列上的一对引物进行PCR鉴定,结果表明:其基因组中没有T-DNA插入.遗传分析表明这两株雄性不育突变体由同一单个隐性基因控制,引起不育的主要原因是从花药发育的第8期开始,小孢子细胞质内容物逐渐减少直至消失,到花药发育的第12期,药室内的小孢子只剩下一个花粉壁空壳,故该突变体命名为opw(only pollen wall).利用图位克隆的方法对OPW基因进行了定位,结果表明OPW基因位于第二条染色体上分子标记T28M21和T3G21之间的12 kb区间内,该区间内一共有21个基因注释.通过克隆区间内的基因并测序发现opw-1突变体基因组中At2g40140基因编码序列的外显子在第289和第290个碱基之间插入了一个A碱基,而opw-2突变体基因组中At2g40140基因编码序列的外显子在第412和第413个碱基之间插入了一个T碱基,造成的编码序列移码使第424至第426碱基成为终止密码子,故At2g40140是编码OPW的候选基因.

Pollen wall pattern in Arabidopsis

The pollen wall is a solid and variously sculptured structure. This pattern is determined inside a tetrad. During meiosis, the callose wall is formed outside of the meiocyte/microspore to form a tetrad. Then, primexine is deposited between the callose wall and the microspore plasma membrane which will become undulated. The sporopollenin deposits on top of the undulated membrane and develops into the pollen wall pattern, while the callose wall is gradually degraded. In recent years, much progress has been made in the study of pollen wall pattern formation, at both molecular and genetic levels. In this review,we summarize these achievements mainly in Arabidopsis.

Ultrastructure analysis reveals sporopollenin deposition and nexine formation at early stage of pollen wall development in Arabidopsis

In angiosperm, pollen wall formation is a critical step for male gametophyte development. Pollen wall constitutes of the outer layer exine and the inner layer intine. Exine is further divided into sexine and nexine. In Arabidopsis, the general process of pollen wall formation has been reported. However, the nexine formation has not been revealed. Here, we observed the process of pollen wall formation in Arabidopsis thaliana using transmission electron microscope. After callose wall is formed, the primexine is present between plasma membrane and the callose layer in the tetrad. With plasma membrane undulation, sporopollenin precursors accumulated on the peak of undulated membrane which is further developed into probacula. The primexine determines plasma membrane undulation and sporopollenin accumulation based on previous analysis of an undulation-deficient mutant. Some materials obviously different from sporopollenin are filled between the primexine and plasma membrane. These materials cover all the surface of plasma membrane and gradually develop into nexine. After microspore is released from tetrad, the nexine layer is formed and the probacula is further developed into sexine with continued accumulation of sporopollenin. Based on these observations, we proposed a developmental model of early pollen wall formation.

PECTATE LYASE-LIKE10 is associated with pollen wall development in Brassica campestris

PECTATE LYASE‐LIKE10（PLL10） was previously identified as one of the differentially expressed genes both in microspores during the late pollen developmental stages and in pistils during the fertilization process in Chinese cabbage（Brassica campestris ssp. chinensis）. Here, antisense‐RNA was used to study the functions of BcPLL10 in Chinese cabbage. Abnormal pollen was identified in the transgenic lines（bcpll10‐4, ‐5, and ‐6）. In fertilization experiments, fewer seeds were harvested when the antisense‐RNA lines were used as pollen donor. In vivo and in vitro pollen germination assays less germinated pollen tubes were observed in bcpll10 lines. Scanning electron microscopy observation verified that the tryphine materials were over accumulated around the pollen surface and sticked them together in bcpll10.Moreover, transmission electron microscopy observation revealed that the internal endintine was overdeveloped and predominantly occupied the intine, and disturbed thenormal proportional distribution of the two layers in the non‐germinal furrow region; and no obvious demarcation existed between them in the germinal furrow region in the bcpll10 pollen. Collectively, this study presented a novel PLL gene that played an important role during the pollen wall development in B. campestris, which may also possess potential importance for male sterility usage in agriculture.

OsLTP47 may function in a lipid transfer relay essential for pollen wall development in rice

In plants,lipid transfer proteins(LTPs)transport pollen wall constituents from the tapetum to the exine,a process essential for pollen wall development.However,the functional cooperation of different LTPs in pollen wall development is not well understood.In this study,we have identified and characterized a grassspecific LTP gene,Os LTP47,an important regulator of pollen wall formation in rice(Oryza sativa).Os LTP47 encodes a membrane-localized LTP and in vitro lipid-binding assays confirms that Os LTP47 has lipidbinding activity.Dysfunction of Os LTP47 causes disordered lipid metabolism and defective pollen walls,leading to male sterility.Yeast two-hybrid and pull-down assays reveal that Os LTP47 physically interacts with another LTP,Os C6.These findings suggest that the plasma membrane-localized Os LTP47 may function as a mediator in a lipid transfer relay through association with cytosolic and/or locular Os C6 for pollen wall development and that various LTPs may function in a coordinated manner to transport lipid molecules during pollen wall development.

酶法破壁提取蜂花粉功能成分的工艺优化及抗氧化活性分析

目的探索酶解结合超声和碱解从而高效破壁和提取蜂花粉中的活性成分。方法采用扫描电镜对蜂花粉微观结构进行分析,并使用高效液相色谱法分析酚类物质的组成和含量变化。结果经12.5%碱性蛋白酶酶解1 h后,蜂花粉酶解上清液的总酚含量、总黄酮含量和2,2’-联氮-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐[2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium salt,ABTS]阳离子自由基清除能力最高,分别为(14.14±0.23)mg GAE/(g·dw),(2.34±0.04)mg RE/(g·dw)和(167.06±1.39)μmol TE/(g·dw)。将酶解辅以超声协同0.3 mol/L NaOH处理后,总酚、总黄酮、ABTS阳离子自由基清除能力及铁离子还原能力相比未处理花粉分别提高了191%、39%、69%和162%。扫描电镜结果显示,碱处理酶解后花粉细胞壁破碎程度显著增加,花粉内含物质从裂口处向外溢出,与理化指标趋势一致。最佳工艺条件为0.3mol/LNaOH超声-碱处理1h,调节pH至7,12.5%的碱性蛋白酶于50℃下酶解1 h。结论超声碱处理可辅助酶解实现蜂花粉的高效破壁,同时小肽、多酚、黄酮等营养成分也进一步溶出,促进了蜂花粉的高值化利用。

紫纹兜兰的花形态和双雄蕊花药发育及分类学意义

兜兰属的分类系统学争议较大,亟待更多资料澄清。该文利用体式解剖镜和石蜡切片技术观察了紫纹兜兰的花结构和双雄蕊花药发育特征。结果表明:(1)花形态特征支持将紫纹兜兰放置于兜兰亚属的单花斑叶组,包括萼片具脉纹、花瓣长圆形带黑褐斑,唇瓣具直立的耳状结构,退化雄蕊新月形等。(2)花药原基分化出一对侧生并列的药室,花药室中央分化出一条不完全贯穿的不育隔膜组织,将其分化为两个小孢子囊。在花药成熟时,不育隔膜组织被降解吸收,两个花粉囊通过次生融合形成一枚马鞍形的黏性花粉团。(3)发育完整的花药壁有4层,由外到内分别为表皮、药室内壁、中层和绒毡层,符合单子叶型花药壁。花药壁具绒毡层和内绒毡层,均为2核。在2-细胞花粉时期,中层和绒毡层发生降解,药室内壁发生纤维状加厚。(4)小孢子母细胞经同时型胞质分裂形成不同排列方式的小孢子四分体,包括正四面体、左右对称和十字交叉型,并且同一药室的小孢子母细胞减数分裂不同步现象明显。(5)在雄配子体发育阶段,小孢子或保持在四分体内或从四分体中游离出来,经有丝分裂发育为2-细胞型花粉,形成具有黏性的四合花粉或花粉粒。基于现有资料,该文比较分析了紫纹兜兰不完全贯穿的不育隔膜组织、单子叶型花药壁、具2核的绒毡层和内绒毡层、同时型胞质分裂和黏性花药等特征的分类学意义,为理解兜兰属及杓兰亚科的分类学和保护生物学提供新资料。

药用野生稻(Oryza officinalis Wall)和转bar基因水稻(Oryza sativa L.)花粉杂交的基因漂移

采用生殖生物学方法研究了药用野生稻和转bar基因水稻花粉杂交的基因漂移。结果表明 ,供试水稻花粉在药用野生稻柱头上的萌发生长与药用野生稻自花授粉花粉的萌发生长有一定差异 ,表现在穿过柱头的花粉粒百分率及内容物释放和正在凝缩、释放的花粉粒百分率较少。虽然转基因水稻花粉能在药用野生稻柱头上正常萌发生长 ,并能释放内容物 ,但杂交后结实率为 0 ,表明转基因水稻和药用野生稻杂交不亲和。他们的不亲和性不是在花粉的萌发和穿过柱头这一阶段 ,具体原因有待进一步研究。在本实验条件下转基因水稻和药用野生稻没有发生成功的基因交流。

ABCG转运蛋白参与植物雄性育性调控的研究进展

利用雄性不育系制种是利用杂种优势的最有效的途径,尤其对小麦(Triticum aestivum L.)、水稻(Oryza sativa L.)等自花授粉作物的杂种优势利用具有十分重要的意义。ABCG(ATP-binding cassette G transporter)转运蛋白属于ABC(ATP-binding cassette transporter)转运蛋白中的一个大亚族,参与植物各种生物过程,包括调控雄性育性。本文概述了ABC转运蛋白的特点与分类,并系统阐述了ABCG转运蛋白参与调控植物花粉壁和花药角质层发育的研究现状,同时也对ABCG转运蛋白目前研究所存在的问题提出了建议,并对后续的研究进行了展望。

云南松松花粉中总氨基酸的含量测定及超声破壁工艺优选

目的:优选云南松松花粉超声破壁工艺,并建立其总氨基酸的含量测定方法。方法:以天冬氨酸作为对照,采用分光光度法测定云南松松花粉中总氨基酸含量;通过单因素试验和正交试验考察超声破壁最佳试验条件。结果:线性方程为A=92.143C-0.0603,r=0.9995,天冬氨酸的质量浓度在2.0~10.0μg·mL^(-1)时与吸光度成良好的线性关系,平均回收率高达99.6%,相对标准偏差为1.85%(n=6);超声波破壁松花粉的最佳工艺为物料量5 g、超声功率700 W、超声时间40 min,破壁率可达98.0%。结论:建立的云南松松花粉中总氨基酸含量的测定方法准确性好;优选的破壁方法稳定可行。

Research Progress of Cell Wall Breaking Technology and Its Application in Eucommia ulmoides Male Flower Tea

Eucommia ulmoides male flowers are rich in secondary metabolite,which have anti-tumor,sedative hypnotic,hypotensive,hypolipidemic,anti-fatigue,bacteriostatic,antioxidant and anti-aging effects.The conventional processing of E.ulmoides male flowers leads to the loss of nutrients and active ingredients.In recent years,cell wall breaking technology has been developed and utilized in various fields such as traditional Chinese medicine,good,chemical industry and biology.In order to promote the development of the E.ulmoides industry,the cell wall breaking technology and its characteristics are reviewed,and the application advantages of the cell wall breaking technology in the male flowers of E.ulmoides are discussed,and the prospect of the cell wall breaking of E.ulmoides is proposed in this article.

Variations in Carbohydrate Content and Sucrose-Metabolizing Enzymes in Tomato (<i>Solanum lycopersicum</i>L.) Stamen Parts during Pollen Maturation

The formation of mature and fertile pollen grains, taking place inside the anther, depends on supply of assimilates, in the form of sucrose, provided mainly by the leaves. Data is limited, however, with respect to the understanding of sucrose metabolism in microspores and the supporting tissues. The aims of the present work were to 1) follow the changes in total and relative concentrations of sucrose, glucose, fructose and starch in the stamen parts and microspores up until anthesis, 2) follow the activities of sucrose-metabolism-related enzymes, in the anther walls fraction and microspores of the crop plant tomato. Sucrose was found to be partially cleaved in the filament, decreasing by more than twofold in the anther wall layers and the locular fluid, and to accumulate in the mature pollen grains, constituting 80% of total soluble sugars. Thus, sucrose was both the starting sugar, supporting microspore development, and the main carbohydrate accumulated at the end of the pollen-development program. The major invertase found to be active in both the anther wall layers and in maturing microspores was cell-wall-bound invertase. High fructokinase 2 and sucrose phosphate synthase activities during pollen maturation coincided with sucrose accumulation. The potential importance of sucrose accumulation during pollen dehydration phase and germination is discussed.

三种核桃花粉破壁方法比较

比较了温差破壁、微波-酶解破壁、超声波破壁对核桃花粉的破壁率、VC含量、抗氧化总能力的影响,结果表明:微波-酶解所得的花粉有较高破壁率、V_(C)含量、较大抗氧化总能力。

破壁处理对松花粉营养成分和理化性质的影响

为探究破壁处理对松花粉营养成分及相关理化特性的影响,对原松花粉和破壁松花粉中营养成分、持水力、持油力、阳离子交换能力、胆固醇吸附能力、亚硝酸盐清除能力和金属离子吸附能力等进行测定和分析。结果表明:经破壁处理后,松花粉粗脂肪和可溶性总糖含量明显升高;总黄酮、总酚和总三萜含量分别增加了18.6%、30.2%和29.6%。松花粉的持水力和持油力、阳离子交换能力和亚硝酸盐清除能力明显增强,胆固醇吸附能力无明显变化,对Cu^(2+)吸附能力增强,而对Cd^(2+)和Pb^(2+)吸附能力减弱。松花粉经过破壁处理后其主要营养和功能成分溶出较多、含量明显增加,大部分理化性质明显改善。

通过遗传转化Rfo获得青花菜Ogura CMS恢复系

【目的】将来自萝卜的胞质不育恢复基因Rfo导入到青花菜Ogura细胞质雄性不育(cytoplasmic male sterility,CMS)主栽商业品种‘耐寒优秀’(命名为SFB45)中,获得Ogura CMS恢复系,打破Ogura CMS材料在生产上无法再利用的现状,为优异种质的创制和改良提供新途径。【方法】利用基因合成的方法克隆萝卜Rfo的CDS及其启动子序列,构建表达载体pRfo::Rfo。通过农杆菌介导的遗传转化方法侵染SFB45的具柄子叶和下胚轴,进行再生、抗性苗筛选和PCR鉴定获得阳性植株,开花期观察转基因植株的育性恢复情况;用亚历山大染液分析对照组及阳性株的花粉活力;分别提取对照组及阳性株<3 mm和>3 mm花蕾的RNA并反转录成单链cDNA,设计特异引物,利用RT-PCR分析Rfo及绒毡层和花粉壁发育相关基因在SFB45及对应的育性恢复材料花蕾中的表达差异。【结果】通过遗传转化共获得10个阳性株系,其中8个株系育性得到不同程度的恢复,花粉活力为84.2%—90.4%。RT-PCR分析结果表明,育性恢复植株花蕾中Rfo表达,绒毡层发育关键调节因子DYT1和TDF1及花粉壁主要成分孢粉素合成所必需的基因ACOS5在育性恢复植株<3 mm花蕾中上调表达。绒毡层降解相关基因AMS、四分体胼胝质壁及花粉外壁发育所必需的基因CalS5和CYP703在育性恢复植株>3 mm花蕾中上调表达。分析阳性株系R-1、R-3和R-6自交及杂交后代发现转入的Rfo能稳定遗传,符合孟德尔遗传。【结论】通过遗传转化Rfo获得了优良青花菜Ogura CMS商品种SFB45的育性恢复系,且Rfo的转入使绒毡层及花粉壁发育相关基因恢复正常表达。

凤丹牡丹花粉和花药壁营养成分分析及评价

为探明凤丹牡丹花粉、花药壁的营养成分,从而更好地发挥其营养价值,本研究采用凯氏定氮法、电感耦合等离子体发射光谱、高效液相色谱、氨基酸分析仪等对凤丹花粉和花药壁蛋白质、矿物质元素、维生素C、水解氨基酸及游离氨基酸进行了系统地分析和评价。结果表明,凤丹花粉中蛋白质含量高达40.68%(初开期),水解氨基酸总量为355.65 mg·g^(-1)(初开期),花药壁中蛋白质和水解氨基酸的总量虽仅为花粉的一半,但二者必需氨基酸与总氨基酸的比值均为40%左右,属优质蛋白来源。36种游离氨基酸检测结果显示,花药壁中总游离氨基酸含量稍高,但花粉中必需氨基酸、蛋白氨基酸及鲜甜氨基酸含量更高。矿物质元素分析结果表明,凤丹花粉、花药壁含有丰富的常量和微量元素,尤以K、Ca、Mg、Fe、Zn等元素含量较高,是开发高钾低钠和补铁补锌的佳品。Vc测定结果显示,凤丹花粉和花药壁中Vc含量较高,可作为Vc补充剂。同时考察了凤丹花粉、花药壁不同采收时期及破壁对检测成分含量的影响,结果显示二者更适合在初开期采收,且破壁对花粉、花药壁营养成分检测无显著影响。综上,凤丹花粉、花药壁含有丰富的营养成分,本研究将为其资源开发和利用奠定基础。

破壁油菜花粉总黄酮含量的测定及体外抗氧化作用的研究

采用NaNO2-Al(NO3)3显色法测定破壁油菜花总黄酮的含量;采用DPPH自由基清除能力、羟自由基清除能力及超氧阴离子自由基清除能力检测破壁油菜花的抗氧化活性。研究发现破壁油菜花粉总黄酮含量为94.16 mg/g;同时破壁油菜花粉会剂量依耐性的分别在浓度为20 mg/mL、10 mg/mL、5 mg/mL使得DPPH自由基、羟自由基、超氧阴离子自由基清除能力达到最大。此研究表明破壁油菜花粉中含有丰富的黄酮类化合物,表现出较好的体外抗氧化作用为其应用提供了理论依据。

玉米花粉混合破壁法的研究

玉米花粉含有丰富的营养物质,不破壁的花粉不易被人体很好地消化吸收。采用温差法、超声波法、酶法和混合法四种方法对玉米花粉进行破壁处理。实验采用L9(33)正交设计。温差法破壁的最优条件是料液比为1:5,温差为90℃,水浴时间为1h。酶法破壁的最优方案是料液比为2%,水浴温度为55℃,水浴时间为12h。混合法是先利用温差法进行破壁,再用超声波法与酶法进行破壁处理。四种方法破壁效果分别为:超声波法31.2%,温差法为46.3%,酶法80.4%,混合法98.6%。酶法和混合法的破壁率极显著地高于超声波法和温差法。混合破壁法效果最好,破壁率达98%以上,具有良好的利用前景。

玉米花粉的破壁处理及营养学研究

我国有极其丰富的玉米花粉资源，至今未被充分利用，为了合理地开发这一营养宝库，我们对玉米花粉进行了破壁工艺研究，并对破壁与未破壁的玉米花粉进行了全面营养成分分析、营养评价等实验研究。结果表明采用急变温差与机械法相结合的破壁方法，工艺简单，高倍镜及电镜检测证明，破壁率达９１％，而且破壁后的花粉含有更丰富的游离氨基酸、维生素、微量元素等。营养评价证明破壁花粉的蛋白质功效比值（ＰＥＲ）、表现消化率（ＡＤ）、和蛋白质净利用率（ＮＰＵ）均显著优于未破壁花粉。本研究为科学地开发利用我国的玉米花粉资源提供了依据。