梅花花器官cDNA文库的构建及其PmAP_3、PmPI、PmAG基因的克隆
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摘要
梅花(Prunus mume Sieb.et.Zucc.)是我国传统名花,品种繁多,蕴含丰富的遗传信息。梅幼年期较长,杂交育种进展缓慢,致使梅花重要的观赏性状和农艺性状研究严重滞后。通过梅花花器官cDNA文库的构建以及花发育相关基因的克隆,研究梅花花发育相关理论,为加速梅花育种及性状改良提供理论依据。主要结果如下:
1.用Biozol法、SDS法和CTAB法三种方法提取梅花组织总RNA。Biozol法操作简单,实验过程短,但总RNA完整性被破坏。SDS法提取的总RNA有降解,并且还有少量DNA污染。CTAB法能够提取高质量总RNA,总RNA完整性较好,没有降解,28s RNA和18s RNA的比例接近2∶1。
2.构建梅花花器官cDNA文库,随机挑选克隆测序获得了239条高质量EST序列。构建的原始文库滴度为5.41×10~6 pfu/ml,重组率为97.29%,所得原始文库滴度及重组率符合建库要求;原始文库经扩增后共得到约200ml库液,扩增文库的滴度1.31×10~(10)pfu/ml,扩增文库滴度符合预期数量级;文库插入片段在500bp-1200bp之间,平均为800bp左右,插入片段大小基本符合要求,文库质量比较高。利用MIPS数据库对239条EST序列比对。结果175条序列和拟南芥已知基因相似性很高,占73.22%;35条序列与拟南芥基因功能尚未确定的假定蛋白或未知蛋白同源性很高,占14.64%;29条序列和拟南芥已知基因没有同源性,占12.13%。3.通过同源序列法,利用RACE和TAIL-PCR从梅花花器官中成功克隆到PmAP_3、PmPI、PmAG三个全长基因。PmAP_3的全长为975bp,包含一个完整的读码框,编码202个氨基酸。将PmAP_3基因推测的氨基酸序列在NCBI上进行Blastp搜索发现,PmAP_3蛋白是AP_3-like蛋白。PmPI的全长为888bp,包含一个完整的读码框,编码210个氨基酸。将PmPI基因推测的氨基酸序列在NCBI上进行Blastp搜索发现,PmPI推测蛋白是PI-like蛋白。PmAG的全长为1171bp,包含一个完整的读码框,编码243个氨基酸。将PmAG基因推测的氨基酸序列在NCBI上进行Blastp搜索发现,PmAG推测蛋白是AG-like蛋白。
4.成功构建PmAP_3、PmPI基因超量表达载体,为进一步研究B类基因的功能打下基础。
Mei flower(Prunus mume Sieb.et.Zucc),an traditional famous flower,has numerous cultivars and contains a wealth of genetic informations.It has a longer childhood and slowly development of cross breeding,which resulted in lagging study of important ornamental and agronomic traits.In order to clarify the mechanism of Mei floral organ development and accelerate its breeding program with improved genetic characteristics,the floral organ cDNA library were constructed and related genes in flower development were cloned,
1.Comparing the quality of Mei total RNA extracted by the Biozol、SDS and CTAB methods resepctively,the results showed that the procedure of Biozol reagent extraction was simpler and time shorter but integrity of RNA was destroyed.By the means of the SDS,total RNA was partly degradation in the extraction process and there was a small amount of genomic DNA contamination.Quality and integrity of total RNA were best by the CTAB method and there was no significant degradation.Ratio of Concentration of 28s RNA and 18s RNA are close to 2:1.
2.cDNA library of Mei floral organs was successfully constructed and obtained 239 EST by sequencing of random selecting clones.The titer of primary library was 5.41×10~6 pfu/ml and recombinant percent accounted for 97.29%,which reached the demands.The primary library was amplified and 200ml liquid containing bacterium was collected.The titer of the amplified library was 1.31×10~(10) pfu/ml,which got the expected magnitude.The size of insert fragments basically ranged from 0.5 kb to 1.2 kb with average of 0.8 kb.The size of insert fragments was consonant with the demands.The results indicated that it was a relatively high quality library.By blast of the sequences in MIPS database,the results were as follows:175 EST sequences of 73.22%showed high homologue with the genes of Arabidopsis thaliana,whose protein function were classified; 35 EST sequences of 14.64%matched to the genes ofArabidopsis thaliana,whose protein function were unclassified;29 EST sequences of 12.13%had no identity sequence in MIPS.
3.Three full-length gene PmAP_3,PmPI and PmAG from Mei using RACE and TAIL-PCR through homologous cloning.Full length of PmAP_3 was 975bp,containing a complete ORF and encoding 202 amino acids.Through BLASTP Research in NCBI using PmAP_3 amino acid sequence,we found that the deduced protein of PmAP_3 was AP_3-like protein.Full length of PmPI was 888bp,which contained a complete ORF,encoding 210 amino acids.BLASTP results showed that the deduced protein of PmPI was PI-like protein.PmAG was 1171 bp,containing a complete ORF,and encoded 243 amino acids. BLASTP research found that the deduced protein of PmAG was AG-like protein.
4.Over-expression vectors of PmAP_3,PmPI were successfully constructed for the further function study of B class genes.
1.用Biozol法、SDS法和CTAB法三种方法提取梅花组织总RNA。Biozol法操作简单,实验过程短,但总RNA完整性被破坏。SDS法提取的总RNA有降解,并且还有少量DNA污染。CTAB法能够提取高质量总RNA,总RNA完整性较好,没有降解,28s RNA和18s RNA的比例接近2∶1。
2.构建梅花花器官cDNA文库,随机挑选克隆测序获得了239条高质量EST序列。构建的原始文库滴度为5.41×10~6 pfu/ml,重组率为97.29%,所得原始文库滴度及重组率符合建库要求;原始文库经扩增后共得到约200ml库液,扩增文库的滴度1.31×10~(10)pfu/ml,扩增文库滴度符合预期数量级;文库插入片段在500bp-1200bp之间,平均为800bp左右,插入片段大小基本符合要求,文库质量比较高。利用MIPS数据库对239条EST序列比对。结果175条序列和拟南芥已知基因相似性很高,占73.22%;35条序列与拟南芥基因功能尚未确定的假定蛋白或未知蛋白同源性很高,占14.64%;29条序列和拟南芥已知基因没有同源性,占12.13%。3.通过同源序列法,利用RACE和TAIL-PCR从梅花花器官中成功克隆到PmAP_3、PmPI、PmAG三个全长基因。PmAP_3的全长为975bp,包含一个完整的读码框,编码202个氨基酸。将PmAP_3基因推测的氨基酸序列在NCBI上进行Blastp搜索发现,PmAP_3蛋白是AP_3-like蛋白。PmPI的全长为888bp,包含一个完整的读码框,编码210个氨基酸。将PmPI基因推测的氨基酸序列在NCBI上进行Blastp搜索发现,PmPI推测蛋白是PI-like蛋白。PmAG的全长为1171bp,包含一个完整的读码框,编码243个氨基酸。将PmAG基因推测的氨基酸序列在NCBI上进行Blastp搜索发现,PmAG推测蛋白是AG-like蛋白。
4.成功构建PmAP_3、PmPI基因超量表达载体,为进一步研究B类基因的功能打下基础。
Mei flower(Prunus mume Sieb.et.Zucc),an traditional famous flower,has numerous cultivars and contains a wealth of genetic informations.It has a longer childhood and slowly development of cross breeding,which resulted in lagging study of important ornamental and agronomic traits.In order to clarify the mechanism of Mei floral organ development and accelerate its breeding program with improved genetic characteristics,the floral organ cDNA library were constructed and related genes in flower development were cloned,
1.Comparing the quality of Mei total RNA extracted by the Biozol、SDS and CTAB methods resepctively,the results showed that the procedure of Biozol reagent extraction was simpler and time shorter but integrity of RNA was destroyed.By the means of the SDS,total RNA was partly degradation in the extraction process and there was a small amount of genomic DNA contamination.Quality and integrity of total RNA were best by the CTAB method and there was no significant degradation.Ratio of Concentration of 28s RNA and 18s RNA are close to 2:1.
2.cDNA library of Mei floral organs was successfully constructed and obtained 239 EST by sequencing of random selecting clones.The titer of primary library was 5.41×10~6 pfu/ml and recombinant percent accounted for 97.29%,which reached the demands.The primary library was amplified and 200ml liquid containing bacterium was collected.The titer of the amplified library was 1.31×10~(10) pfu/ml,which got the expected magnitude.The size of insert fragments basically ranged from 0.5 kb to 1.2 kb with average of 0.8 kb.The size of insert fragments was consonant with the demands.The results indicated that it was a relatively high quality library.By blast of the sequences in MIPS database,the results were as follows:175 EST sequences of 73.22%showed high homologue with the genes of Arabidopsis thaliana,whose protein function were classified; 35 EST sequences of 14.64%matched to the genes ofArabidopsis thaliana,whose protein function were unclassified;29 EST sequences of 12.13%had no identity sequence in MIPS.
3.Three full-length gene PmAP_3,PmPI and PmAG from Mei using RACE and TAIL-PCR through homologous cloning.Full length of PmAP_3 was 975bp,containing a complete ORF and encoding 202 amino acids.Through BLASTP Research in NCBI using PmAP_3 amino acid sequence,we found that the deduced protein of PmAP_3 was AP_3-like protein.Full length of PmPI was 888bp,which contained a complete ORF,encoding 210 amino acids.BLASTP results showed that the deduced protein of PmPI was PI-like protein.PmAG was 1171 bp,containing a complete ORF,and encoded 243 amino acids. BLASTP research found that the deduced protein of PmAG was AG-like protein.
4.Over-expression vectors of PmAP_3,PmPI were successfully constructed for the further function study of B class genes.
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