| Chinese mustard crops (Brassica juncea Coss.) are one traditional Chinese vegetable mainly for pickling, which are wide-spreadly cultivated in Zhejiang and Sichuan provinces. It is of great economic importance and its pickled products are favored by consumers in China and Southeastern Asian countries due to its rich nutritional components, such as, vitamin, amino acid, antioxidant and ploy-unsaturated fatty acid. However, virus and poor-quality are main problems in agricultural practices. The utilization of heterosis usually may bring sunshine to such practical problems. However, mustard crop is a self-pollinated crop with up to 30 % crossed pollination. To date, all cultivars of mustard crops in China are fixed ones and no F1 hybrids have ever been released. Undoubtedly, cytoplasmic male sterility is an alternative way to produce hybrids of Chinese mustard crops according to its sterile rate of 100%. Stem mustard (Brassica juncea var. tumida Tsen et Lee) cms lines were synthesized by distant hybridizations and subsequent backcrossings. The progenies of advanced backcrossed generation of BC13 and its corresponding fertile maintainer lines were used as the source of sterile and fertile cytoplasms. The plants in fertile maintainer line was concomitantly self-crossed and used as recurrent parents. Genetically, both cms and its fertile maintainer lines could be treated as near isogenic lines by the 13th backcrossed generation, and favorable for comparative analysis of molecular aspects. They were named after orf220 type male-sterile cytoplasm. Unfortunately, there was no restored line to be naturally found for this cms line.Cytoplasmic male sterility (cms), the maternally inherited character of failure to produce viable pollens, was existent in many plants and widely applicable for heterosis utilization. It is generally believed that cms is frequently associated with dysfunction of mitochondria, which is caused by chimeric open reading frames (orf), post-transcriptional regulations or expressions of specific peptides. Investigations for molecular mechanism of cms might elucidate the interactions between mitochondrion and nucleus and reproductive development in higher plants as well, meanwhile, it could also offers the theoretical basis for molecular operation of mitochondrial genome, creating new cms germplasm through gene engineering and shortening the course of breeding. In our investigations, we try to find the cms-associated genes and the molecular mechanism of cms in stem mustard through comparative research. The results are shown as the followings:1 CMS-associated orf220 gene was isolated from cms stem mustard usinghomological cloning (Genebank Accession No. AY208898). Or/220 gene was deduced to encode one membrane-associated protein and had homology with or/222, or/224 and orfl38 genes found in Brassica species showing 74%, 76% and 38% similarity respectively. Meanwhile, its N-terminus shared higher similarity in amino acids constituents with COXIII protein from Oenothera berteriana, ATP8 protein from radish and ORFB protein from sunflower. In cms stem mustard, or/220 gene was indicated to be single copy and expressed in root, leave, bud and flowers.2 Mitochondrial atpA genes were observed to be numerous differences between cms and its maintainer lines of stem mustard by comparative research. In its maintainer line, atpA gene was 2269 bp sizes and deduced to encode 507 amino acids with typical and activity-necessary domain of ATPase complex. Meanwhile, atpA gene of its maintainer line was indicated of high conservation with that from other species, even 90% homology with atpA gene of Oryza sativa in terms of amino acid constituents, and had nearer phylogenetic relationship with that from Cruciferae. However, in cms line, atpA gene was 2618 bp sizes and deduced to encode 429 amino acids with one 383 bp sized fragment insertion at 5'-UTR (Genebank Accession No. AY211266, AY211265). Meanwhile, atpA genes had same deduced promoter sequence, transcriptional initiation sites and ribosome binding sites between cms and its maintainer lines. Interestingly, atpA gene had significant differences at 3'-flanking region between cms and its maintainer lines using Southern blotting. In addition, atpA gene was transcriptional regulated by its 3'-flanking region in cms line by Northern blotting.3 Mitochondrial nad2 genes were observed to be numerous differences between cms and its maintainer lines of stem mustard by comparative research. One specific nad2-CMSF was cloned from cms line, which was shown to be 297 bp sizes and of high homology with partial sequences of orfB in cms radish, orfB in cms sunflower, atp6 gene in Brassica pol cms, nad5 gene in Brassica nap cms, orjB-CMS in carrot cms and or/220 gene in cms stem mustard. Meanwhile, nad2-CMSF had three copies in cms line and only one copy in its maintainer line by Southern blotting, in addition, two transcripts in cms line and one transcript for its maintainer line by Northern blotting. So, it was deduced that nad2-CMSF was partially composed of mitochondrial nad2 gene and others originned from cms-associated gene. So, we deduced that nad2-CMSF would be the rearrangements loci in mitochondria and could impact on mitochondrial normal function, which might be a causal factor leading to male sterility in this type cms stem mustard.4 RNA editing for mitochondrial atp9 gene was indicate of significant differences between cms and its maintainer lines of stem mustard. There were four editing sites for mitochondrial atp9 gene according to its normal editing sites in mustard, of which 3 sites occurred as C-to-U changes and one as U-to-C change (Genebank Accession No. AY847286). As a result, the hydrophobicity of deduced atp9 protein was reduced according to the conversions at 17th, 45th and 64th positions, and the conservation of deduced atp9 protein was enhanced by the changes at 56th position. Loss of specific editing site for atp9 gene was observed in juvenile roots, senile roots and floret buds of cms line. Comparatively, complete RNA editings of atp9 gene were retained in juvenile roots, juvenile leaves and floret buds in its maintainer line, however, loss of specific editing site of atp9 gene occurred at senile roots and senile leaves. These observations allow us to produce a hypothesis underlying cms and organs senescence that dysfunction of specific mitochondrial genes arising of RNA editing could probably be candidate factors triggering cms and organs senescence through unknown cross-talk pathways during development.5 Nuclear homeotic genes, APETALA3 (BjAP3, Genebank, DQ060332), PISTILLATA (BjPI, Genebank, DQ060333i and AGAMOUS (BjAG, Genebank, DQ060334), were isolated from stem mustard, in which these three nuclear homeotic genes shared high conserved MADS-box domain and relative conserved K-box domain, in addition, the PI motif being solely present in PISTILLATA was observed at the end of BjPI gene. The expression patterns of these three genes were altered in the cms stem mustard with BjAP3 and BjAG genes being greatly decreased in expression abundance at the third whorl of floral organ and being appeared unexpectedly at the fourth whorl of floral organ and at the second whorl floral organs respectively. Furthermore, the expression for BjAG gene was enhanced at the fourth whorl of floral organs. Interestingly, the ectopic expressions of BjAP3 in pistils and BjAG in petals were first identified in aberrated flowers of cms stem mustard. With respect to BjPI in cms line, the abundance of transcripts was observed to be reduced at the third whorl of floral organ.6 SPOROCYTELESS (SPL) gene, defined as controlling microspogenesis, was isolated from stem mustard using RT-PCR combined with RACE methods and then named after BjSPL. The full length of cDNA for BjSPL gene was sequenced to be 1595 bp-sized and deduced to encode 313 amino acid, which was shown to be 69% homology with SPL(NZZ) gene from Arabidopsis in terms of amino acid components (Genebank Accession No. DQ358088). Likewise, BjSPL gene was deduced to beencoded one nuclear hydrophilic MADS-like transcriptional factor (TF) protein. Furthermore, BjSPL gene was normally expressed in early and middle developmental stage of buds in fertile stem mustard, however, only an exceeding small quantity of transcription was observed in early developmental stage of buds and no expression in middle developmental stage of buds in cytoplasmic male sterile line of stem mustard. So, these investigations allowed us to deduce that failure of microsporogenesis was controlled by BjSPL gene under the regulation of AG gene in alloplasmic cytoplasmic male sterile of stem mustard.7 Mitochondrial targeted expression vector for or/220 gene, PBI121-ATPP -ORF220, was constructed and transferred into fertile stem mustard and wilt type Arabdopsis mediated by Agrobacterium to confirm the real biology funtion. Orf220 gene was identified to be successfully transferred into fertile stem mustard and wilt type Arabdopsis by PCR check in transgenic plants. The traits and expressions of a series of genes would be observed and investigated in transgenic plants in the near future.8 Mitochondrial inhibitor could induce the abnormal floral organs development and pollen sterility. Combinations of stamen and petal phenotype and a great deal of wizened pollens were observed after being treated by 400umol/L NaN3. Meanwhile, germination of pollens were also severely impacted after being treated by 400umol/L NaN3. Likewise, wizened pollens with different extent were also found after being treated by 12.5> 25 and lOOumol/L oligomycin. In addition, expressions of nuclear homeotic genes {BjAG> BjAP3and BjPI genes) and microsporegenesis gene {BjSPL gene) were induced to be decreased after being treated by mitochondrial inhibitors. Meanwhile, seed of self-pollination is almost zero treated by mitochondrial inhibitor, and it could be recovered to be normal after stopping to be treated. Likewise, seed of nature-pollination is almost normal.9 The intra-specific hybridization and subsequent backcrossings were employed to synthesize cms leaf mustard (Brassica juncea var. multiceps Tsen et Lee). Plants of tuber mustard were taken as cms donor and fertile plants of leaf mustard were used as recurrent parents and resultant maintainers of cms as well. Meanwhile, the biology traits were observed and statistic for the hybridization and backcrossings generations during cms transfer. Series of abnormal stamens, zero self-pollinated seedy and normal seedy with hand-pollinated were shown in cms BC3 generation of leaf mustard. In addition, economic traits and nectary numbers was shown to be gradually recovered to be like its maintainer line of leaf mustard. Meanwhile, severalcms-associated genes were proved to be maternally transmitted and genetically stable in various backcrossed progenies.10 According to the above results, one hypothesis would be made about pathway of interaction between mitochondria and nucleus for cytoplasmic male sterility formation. Mitochondrial specific orf, alterations on mitochondrial energy metabolism-related genes or other changes in mitochondrion could change the rate of mitochondrial metabolism, and then, microspore development gene, its upstream genes and some nuclear homeotic genes would be regulated through signal transduction pathways in cms plant. Finally, pollen sterility and some abnormal stamens were observed in alloplasmic cms plant.
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