花粉发育相关基因BcMF13不同转录本的克隆及分析

在克隆花粉发育相关基因BcMF13时,用RACE技术进行3’端扩增,得到了两个不同长度的3’转录本。与EST库比对,发现与其相似性高的序列都来源于十字花科芸薹属、萝卜属等植物的生殖器官,并且在这些近源属中也出现不同转录本。这些不同转录本的存在说明BcMF13存在剪切异构体,调控过程灵活复杂,反映出该基因具有重要功能。同时,对BcMF13基因的RT-PCR表达研究表明,它只在可育株系中表达,在不育株系中不表达,且主要集中在大花蕾、雄蕊中表达。分析BcMF13推导的蛋白结构,发现其编码的蛋白包含多个生物活性位点。以上结果均表明BcMF13与花粉育性相关,多个剪切异构体的发现说明BcMF13在执行生理功能时较为活跃。     

Morphological and functional characterization of BcMF13 in the antisense-silenced plants of Brassica campestris ssp. chinensis var. parachinensis

The gene Brassica campestris male fertility 13 (BcMF13, GenBank accession number EF158459) was isolated as a reproductive organ-specific gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). It is exclusively expressed in stage four and five flower buds of fertile lines and is most strongly expressed in stamens. Here, we report a functional characterization of this BcMF13 gene in the antisense-silenced plants. The inflorescence of the BcMF13 mutant was compacted with anthers curved outside. The fertility of this mutant was greatly reduced with less than 5 seeds per silique. Under scanning electron microscopy, the mutant demonstrated numerous shriveled pollen grains with deep invaginations. The frequency of normal pollen grains was just 45.34%. The pollen mother cell, the tetrad, and the mature pollen of the BcMF13 mutant were abnormal resulting in the poor pollen vitality. Germination test in vivo suggested BcMF13 delayed the pollen tubes’ extension in the style. All these indicated BcMF13 had a vital role in pollen development of Chinese cabbage.     

BcMF9, a novel polygalacturonase gene, is required for both Brassica campestris intine and exine formation

Background and Aims

The polygalacturonase (PG) gene family has been found to be enriched in pollen of several species; however, little is currently known about the function of the PG gene in pollen development. To investigate the exact role that the PG gene has played in pollen development and about this family in general, one putative PG gene, Brassica campestris Male Fertility 9 (BcMF9), was isolated from Chinese cabbage (Brassica campestris ssp. chinensis, syn. B. rapa ssp. chinensis) and characterized.

Methods

RT-PCR, northern blotting and in situ hybridization were used to analyse the expression pattern of BcMF9, and antisense RNA technology was applied to study the function of this gene.

Key Results

BcMF9 is expressed in particular in the tapetum and microspore during the late stages of pollen development. Antisense RNA transgenic plants that displayed decreased expression of BcMF9 showed pollen morphological defects that resulted in reduced pollen germination efficiency. Transmission electron microscopy revealed that the homogeneous pectic exintine layer of pollen facing the exterior was over-developed and predominantly occupied the intine, reversing the normal proportional distribution of the internal endintine layer and the external exintine in transgenic pollen. Inhibition of BcMF9 also resulted in break-up of the previously formed tectum and baculae from the beginning of the binucleate stage, as a result of premature degradation of tapetum.

Conclusions

Several lines of evidence, including patterns of BcMF9 expression and phenotypic defects, suggest a sporophytic role in exine patterning, and a gametophytic mode of action of BcMF9 in intine formation. BcMF9 might act as a co-ordinator in the late stages of tapetum degeneration, and subsequently in the regulation of wall material secretion and, in turn, exine formation. BcMF9 might also play a role in intine formation, possibly via regulation of the dynamic metabolism of pectin.Key words: Brassica campestris, Chinese cabbage, exine, intine, PG, pollen wall, polygalacturonase, BcMF9     

The polygalacturonase gene BcMF2 from Brassica campestris is associated with intine development

Brassica campestris Male Fertility 2 (BcMF2) is a putative polygalacturonase(PG) gene previously isolated from the flower bud of Chinesecabbage (Brassica campestris L. ssp. chinensis Makino, syn.B. rapa ssp. chinensis). This gene was found to be expressedspecifically in tapetum and pollen after the tetrad stage ofanther development. Antisense RNA technology was used to studythe function of BcMF2 in Chinese cabbage. Scanning and transmissionelectron microscopy revealed that there were deformities inthe transgenic mature pollen grains such as abnormal locationof germinal furrows. In addition, the homogeneous pectic exintinelayer facing the exterior seemed to be overdeveloped and predominantlyoccupied the intine, thus reversing the normal proportionaldistribution of the internal endintine layer and the externalexintine layer. Since it is a continuation of the intine layer,the pollen tube wall could not grow normally. This resultedin the formation of a balloon-like swelling structure in thepollen tube tip in nearly 80% of the transgenic pollen grains.Premature degradation of tapetum was also found in these transgenicplants, which displayed decreased expression of the BcMF2 gene.BcMF2 might therefore encode a new PG with an important rolein pollen wall development, possibly via regulation of pectin'sdynamic metabolism. Key words: Brassica campestris, Brassica rapa, Chinese cabbage, intine, PG, polygalacturonase, pollen wall Received 28 August 2008; Revised 14 October 2008 Accepted 20 October 2008     

花粉发育相关基因BjMF6的分子克隆及其生物信息学分析

根据白菜花粉特异的多聚半乳糖醛酸酶(PG)基因BcMF6的全长序列设计引物,通过PCR直接扩增的方法从茎瘤芥(BrassicajunceaCzern.etCossvar.tumidaTsenetLee)‘浙桐1号’和分蘖芥(Brassicajunceavar.multicepsTsenetLee)‘雪里蕻’中克隆到了BcMF6的同源基因BjMF6t和BjMF6m。BjMF6t和BjMF6m的序列完全相同,故合称BjMF6。该基因与BcMF6在DNA水平的相似性高达99.6%。Blast分析发现它与拟南芥外切PG基因在氨基酸水平上有很高的相似性,并且具有所有PG基因特有的功能结构域和活性位点,推测BjMF6属于PG基因。通过序列分析和系统树构建进一步证明了该基因可能是与花粉发育相关的PG基因。     

The distinct functions of two classical arabinogalactan proteins BcMF8 and BcMF18 during pollen wall development in Brassica campestris

Arabinogalactan proteins (AGPs) are extensively glycosylated hydroxyproline‐rich glycoproteins ubiquitous in all plant tissues and cells. AtAGP6 and AtAGP11, the only two functionally known pollen‐specific classical AGP encoding genes in Arabidopsis, are reported to have redundant functions in microspore development. BcMF18 and BcMF8 isolated from Brassica campestris are the orthologues of AtAGP6 and AtAGP11, respectively. In contrast to the functional redundancy of AtAGP6 and AtAGP11, single‐gene disruption of BcMF8 led to deformed pollen grains with abnormal intine development and ectopic aperture formation in B. campestris. Here, we further explored the action of BcMF18 and its relationship with BcMF8. BcMF18 was specifically expressed in pollen during the late stages of microspore development. Antisense RNA transgenic lines with BcMF18 reduction resulted in aberrant pollen grains with abnormal cellulose distribution, lacking intine, cytoplasm and nuclei. Transgenic plants with repressive expression of both BcMF8 and BcMF18 showed a hybrid phenotype, expressing a mixture of the phenotypes of the single gene knockdown plant lines. In addition, we identified functional diversity between BcMF18/BcMF8 and AtAGP6/AtAGP11, mainly reflected by the specific contribution of BcMF18 and BcMF8 to pollen wall formation. These results suggest that, unlike the orthologous genes AtAGP6 and AtAGP11 in Arabidopsis, BcMF18 and BcMF8 are both integral to pollen biogenesis in B. campestris, acting through independent pathways during microspore development.     

A polygalacturonase inhibitory protein gene (<Emphasis Type="Italic">BcMF19</Emphasis>) expressed during pollen development in Chinese cabbage-pak-choi

The level of polygalacturonase inhibitory protein (PGIP) genes involved in pollen development remains unclear. Characterization of the different PGIP genes that are expressed in pollen is necessary in understanding the similarities and differences of functions between the members of this gene family, as well as the underlying mechanism of pollen development. A gene-encoding putative PGIP, BcMF19 was successfully cloned on a cDNA-amplified fragment length polymorphism fragment after it was found to be up-regulated in the fertile flower buds of Chinese cabbage-pak-choi (Brassica campestris L. ssp. chinensis Makino) genic male sterile AB line (Bajh97-01A/B). The amino acid sequence of BcMF19 possessed the basic feature of PGIPs, containing an N-terminal signal peptide, several potential N-glycosylation sites, two disulfide bridges flanking both the N- and C-terminal regions, and 10 leucine-rich repeat (LRR) consensus sequences. Real-time RT-PCR verified the higher expression of BcMF19 in the fertile flower buds compared to the sterile flower buds. In situ hybridization showed that BcMF19 was exclusively expressed in the tapetal cells and microspores during anther development. These results indicate that BcMF19 is a novel PGIP gene that might be involved in pollen or tapetum development.     

白菜雄性不育相关基因BcMF4基因功能的RNAi验证

BcMF4(Brassica campestris Male Fertility 4)是前一阶段从普通白菜 (Brassica campestris ssp. chinensis var. communis, syn. B. rapa ssp. chinensis var. communis)核雄性不育两用系的可育株中分离到的雄性不育相关基因。本研究根据BcMF4基因的cDNA序列设计两对特异引物, 从普通白菜花蕾cDNA中扩增出两个片断后连接至双元载体pBI12l中, 得到了RNAi (RNA interference) 植物表达栽体pBI-B4R, 并导入了农杆菌LBA4404菌株中; 通过组织培养途径转化菜心(B. campestris ssp. chinensis var. parachinensis), 72.2%的菜心转基因植株中45.8%的花粉为缩小而空瘪的畸形, 而且这些植株的花粉离体萌发率降低至23.7%; Northern杂交显示, 转基因植株的花粉畸形, 是由于BcMF4基因片段的插入使BcMF4基因的表达受到了抑制。结果表明, 采用RNAi技术下调了BcMF4基因的表达, 导致了菜心转基因植株部分花粉的不育, 证明BcMF4基因在普通白菜和菜心等白菜植物的花粉发育中起着重要作用。     

Characterization and functional analysis of a novel PCP gene BcMF5 from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino)

Brassica campestris Male Fertile 5 (BcMF5), a novel member of the pollen coat protein class A (PCP-A) gene family, was identified from Brassica campestris L. ssp. chinensis Makino (Chinese cabbage-pak-choi). Temporal and spatial expression analysis showed that BcMF5 is a late-expressed PCP gene related to the process of determining pollen fertility. Functional analysis by hairpin RNA (hpRNA)-mediated RNA interference also showed that the expression of BcMF5 is inhibited, which resulted in the low germination ability of the pollen and also in an abnormality of the pollen exemplified by a collapsed germination furrow. This demonstrates that the expression of BcMF5 is closely related to the tapetum. Further, the expression profile of the BcMF5 promoter in Arabidopsis was also analyzed. This analysis indicated that the BcMF5 promoter began expression in the early stage of anther development and drove high levels of glucuronidase (GUS) expression in anthers, pollen, and the pollen tube in the late stage of pollen development, but did not drive any expression in petals, sepals, or pistils. Together with the functional analysis, the hypothesis that BcMF5 may have a sporophytic or gametophytic expression pattern is presented.     

BcMF11, a novel non-coding RNA gene from Brassica campestris, is required for pollen development and male fertility

Key message

BcMF11 as a non-coding RNA gene has an essential role in pollen development, and might be useful for regulating the pollen fertility of crops by antisense RNA technology.

Abstract

We previously identified a 828-bp full-length cDNA of BcMF11, a novel pollen-specific non-coding mRNA-like gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino). However, little information is known about the function of BcMF11 in pollen development. To investigate its exact biological roles in pollen development, the BcMF11 cDNA was antisense inhibited in transgenic Chinese cabbage under the control of a tapetum-specific promoter BcA9 and a constitutive promoter CaMV 35S. Antisense RNA transgenic plants displayed decreasing expression of BcMF11 and showed distinct morphological defects. Pollen germination test in vitro and in vivo of the transgenic plants suggested that inhibition of BcMF11 decreased pollen germination efficiency and delayed the pollen tubes’ extension in the style. Under scanning electron microscopy, many shrunken and collapsed pollen grains were detected in the antisense BcMF11 transgenic Chinese cabbage. Further cytological observation revealed abnormal pollen development process in transgenic plants, including delayed degradation of tapetum, asynchronous separation of microspore, and aborted development of pollen grain. These results suggest that BcMF11, as a non-coding RNA, plays an essential role in pollen development and male fertility.     

Isolation and Characterization of an Anther-Specific Polygalacturonase Gene, BcMF16, in Brassica campestris ssp. chinensis

Many genes in the genic male sterile A/B line (Bajh97-01A/B) of Chinese cabbage pak choi (Brassica campestris L. subsp. chinensis Makino) are expressed differentially, and some play critical roles in the formation of pollen walls. In this study, one of these genes, Brassica campestris Male Fertility 16 (BcMF16), has been isolated and characterized. The BcMF16 gene shares approximately 85% nucleotide sequence homology with two exopolygalacturonase (EC3.2.1.67) genes of Arabidopsis thaliana. Cluster analysis of polygalacturonase peptides indicate that BcMF16 belongs to the pollen polygalacturonase clade. Quantitative real-time PCR analysis has revealed that BcMF16 is specifically expressed in reproductive tissues of the fertile line of genic male sterile A/B line of Chinese cabbage pak choi, and that expression levels dramatically increased during later stages of pollen development. In situ hybridization has demonstrated that BcMF16 is specifically and transiently expressed in both tapetum and pollen following microspore separation at the tetrad stage.