CAJ | 학술논문

利用不可产生花粉的突变体作为研究花药和花粉发育的途径，以证实涉及花粉发育的基因及其在基因网络中的作用。研究结果表明，拟南芥中MYB26/MALE STERILE35（ MS35）雄性不育基因会调控药室内壁次生加厚发育，影响其后的花药开裂，并可上调木质素生物合成途径。而At2g47500基因是在芯片分析ms35/myb26突变体中涉及花粉发育表达下调的一个未被鉴定的基因。通过生物信息学分析发现，At2g47500基因是一个与微管发育有关的基因，在整个植株都表达，包括花发育阶段。利用拟南芥基因库筛选该基因中的T-DNA插入突变体，并通过PCR鉴定，得到插入位点在启动子和外显子的纯合突变体。通过表型分析和表达筛选发现，At2g47500突变并未对表现型产生影响，表明T-DNA插入位点在外显子的突变体基因并未在花药中表达；但转录表达分析认为，该基因在花粉里表达，并通过启动子：GUS表达结构得到进一步证实。35S启动子超量表达At2g47500，并未导致其在野生型植物中异位的过量表达；将其转入突变体中，基因表达量恢复，也未有表现差异。说明其对花粉发育的影响不明显。定量PCR分析微管发育其他相关基因，以及花发育中次生壁发育有关基因在突变体中的表达，At2g47500对花粉发育影响不明显。在突变体中，大部分基因也未因其缺失、表达受到明显影响。综上所述，该基因在花药发育中不受ms35/myb26的调控，发挥次要作用，受到主效基因的调控。
이용불가산생화분적돌변체작위연구화약화화분발육적도경，이증실섭급화분발육적기인급기재기인망락중적작용。연구결과표명，의남개중MYB26/MALE STERILE35（ MS35）웅성불육기인회조공약실내벽차생가후발육，영향기후적화약개렬，병가상조목질소생물합성도경。이At2g47500기인시재심편분석ms35/myb26돌변체중섭급화분발육표체하조적일개미피감정적기인。통과생물신식학분석발현，At2g47500기인시일개여미관발육유관적기인，재정개식주도표체，포괄화발육계단。이용의남개기인고사선해기인중적T-DNA삽입돌변체，병통과PCR감정，득도삽입위점재계동자화외현자적순합돌변체。통과표형분석화표체사선발현，At2g47500돌변병미대표현형산생영향，표명T-DNA삽입위점재외현자적돌변체기인병미재화약중표체；단전록표체분석인위，해기인재화분리표체，병통과계동자：GUS표체결구득도진일보증실。35S계동자초량표체At2g47500，병미도치기재야생형식물중이위적과량표체；장기전입돌변체중，기인표체량회복，야미유표현차이。설명기대화분발육적영향불명현。정량PCR분석미관발육기타상관기인，이급화발육중차생벽발육유관기인재돌변체중적표체，At2g47500대화분발육영향불명현。재돌변체중，대부분기인야미인기결실、표체수도명현영향。종상소술，해기인재화약발육중불수ms35/myb26적조공，발휘차요작용，수도주효기인적조공。
We have used mutants that are defective in the production of pollen as a means to study anther and pollen development .This has identified genes that are involved in pollen development and are likely to function in the same network .Previously ,the Arabidopsis thaliana MYB26/MALE STERILE35 ( MS35 ) gene regulated endothe-cial cell development within the anther and acts upstream of the lignin biosynthesis pathway .Gene(At2g47500)re-lated to microtubule identified from microarray analysis of ms35/myb26 mutant showed down-regulated gene expres-sion in mutant were characterized by bioinformatics analysis .Insertional mutants was identified in knockout lines of Arabidopsis GenBank .Homozygous lines screened were then tested for altered expression of the genes of interest by reverse-transcriptase ( RT)-PCR.Lines that show down-regulation were phenotypically characterized for changes in development.RT-PCR expression analysis suggested that the At2g47500 was expressed in the anthers and confirmed using transformed promoter:GUS reporter constructs into wild type Arabidopsis plants by Agrobacterium transforma-tion and analysis.Over-express At2g47500 in wild type plant and compensating in knock out lines ,there was no much effect on general plant growth and specifically on pollen development and only recover expression in mutant . Quantity real time-PCR analysis some genes related to microtubule in mutant ,expression as normal and similar to compare with some genes related to the secondary wall expression in myb26 buds ( immature young and old stage ) . At2 g47500 impact on pollen development is not obvious ,it turns into mutant ,the amount of gene expression ,no per-formance difference .To sum up , the gene plays a secondary role in the anther development , and regulated by the main gene in anther .