| Pomegranate(Punica granatum L.),is a perennial deciduous tree or shrub in the pomegranate family,which is a characteristic fruit tree with both nutritional value and ornamental value.Pomegranate flowers are hermaphroditic flowers,which can be divided into complete flowers and incomplete flowers according to the development of pistils.Complete flowers are trumpet-shaped flowers,well developed and fruitful.Incomplete flowers are cylindric-shaped flowers,which are abnormal in development,easy to fall off.It directly affects the proportion of trumpet-shaped and cylindric-shaped flowers and the yield of pomegranate.Studies have shown that gibberellin is an important factor affecting the formation of rumpet-shaped and cylindric-shaped of pomegranate flowers.In this experiment,the shape index and gibberellin content of pomegranate trumpet-shaped flowers and cylindric-shaped flowers were determined from 'Punica granatum cv.Hongyushizi'.At the same time,the gibberellin biosynthesis pathway key genes(PgKO,PgGA20ox)were cloned and analyzed the expression characteristics of them in trumpet-shaped flowers and cylindric-shaped flowers of pomegranate.After that,the PgKO overexpression vector was constructed to infect Arabidopsis thaliana,obtaining the transgenic plant with PgKO gene.Finally,Gene expression characteristics in different tissues of T3 generation Arabidopsis plants were analyzed.The corresponding experimental results were listed as follow:1.The complete sequence of PgKO and PgGA20ox which are the genes in gibberellin biosynthesis were cloned.The cDNA full-length sequence of PgKO(GenBank accession:MG208017)was 1729 bp encoding 513 bp amino acids with 1542 bp open reading flame and 186 bp non-coding region.The cDNA full-length sequence of PgGA20ox(GenBank accession:MH104888)was 1676 bp encoding 331 bp amino acids with 993 bp open reading flame and 683 bp non-coding region.Sequence alignment with other species showed that the similarities between PgKO and Vnifera cultivate Malus x domestica,Pyrus Communis were 73%,64.56%and 61.28%respectively.At the same time,the similarities between PgGA20ox and Hevea brasiliensis,Citrus sinensis,Fragaria vesca were 65.55%,67.10%and 62.44%respectively.2.The shape indexes of trumpet-shaped flowers and cylindric-shaped flowers of pomegranate were measured,and the content of gibberellin in different organ parts of two flower types was determined by spectrophotometer.The length of trumpet-shaped flowers was longer than that of cylindric-shaped flowers,and the difference between them was not significant.Both trumpet-shaped flowers and cylindric-shaped flowers had the longest petals among six organs,but the calyx was the shortest one in trumpet-shaped flowers,and the style was the shortest one in cylindric-shaped flowers,and the difference between them is significant.The content of gibberellin in the style of trumpet-shaped flowers was highest at 5.83 g/g,followed by ovary,calyx tube,calyx and petal,and the content of gibberellin in anther was lowest at 2.89 g/g.The content of gibberellin in ovary was the highest at 4.32 g/g,followed by style,calyx tube,calyx and petal,and the content of gibberellin in anther was the lowest at 2.45 g/g.The contents of gibberellin trumpet-shaped flowers were higher than those in the cylindric-shaped flowers on the whole.The contents of gibberellin between ovary,petals,calyx tube and style in trumpet-shaped flowers and cylindric-shaped flowers showed significant difference,while the contents of calyx and anther showed no significant difference.3.The expression level of PgKO and PgGA20ox in different organ parts of pomegranate two flower types were analyzed using fluorescence quantification PCR.The expression of PgKO was highest in the style of trumpet-shaped flowers followed by ovary,calyx,calyx tube and petal,and lowest in anther.The expression of PgKO in calyx of cylindric-shaped flowers was the highest,followed by that in style,petal,ovary,calyx tube and anther.The expression of PgKO in calyx of trumpet-shaped flowers and cylindric-shaped flowers showed significant difference,while the expression in other parts of organs showed no significant difference.The expression of PgGA20ox was the highest in calyx tube of trumpet-shaped flowers,followed by style,calyx,petal and ovary,and the expression was the lowest in anther.PgGA20ox had the highest expression in anthers of cylindric-shaped flowers,followed by calyx tube,style,calyx,ovary and petal.The expression of PgGA20ox in calyx tube showed significant difference between trumpet-shaped flowers and cylindric-shaped flowers,while the expression in other organ parts showed no significant difference.The expression of PgKO was consistent with that of gibberellin,and the expression of PgKO in different organ parts was higher than that of PgGA20ox.Gibberellin content and gene expression were significantly different between the two flower types,so gibberellin may be one of the important reasons for the formation of pomegranate trumpet-shaped flowers and cylindric-shaped flowers.4.The relative expression of T3 transgenic Arabidopsis thaliana carried PgKO gene was analyzed by fluorescence quantification PCR.The results showed that the expression levels of PgKO transgenic plants in different tissues were different.And the expression in the flower topped the six which was 4.64 times as much as root,was 2.09 times as much as stem,was 1.13 times as much as leaf and was 3.92 times as much as fruit,respectively.While the expression levels in the leaves was listed at the bottom,followed by fruit,stems and leaves. |
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