多倍体西瓜发育过程中番茄红素变化及关键酶基因表达
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摘要
本研究以7组同基因型不同倍性(二倍体、三倍体、四倍体)西瓜为材料,测定西瓜果实发育过程中番茄红素含量变化和番茄红素合成关键酶基因(八氢番茄红素合成酶PSY-A和番茄红素环化酶LCYB)的转录表达量变化,分析不同倍性西瓜果实发育过程中番茄红素含量与酶基因表达之间的关系；探讨四倍体西瓜不同发育时期的果实、不同组织中番茄红素关键酶基因的时空表达差异；对未成熟、成熟和过熟西瓜果肉细胞的超微结构进行透射电镜的观察,了解不同倍性西瓜发育过程中细胞中有色体的结构变化；从而为多倍体西瓜育种提供实验依据。本试验取得以下主要研究结果：
1.西瓜果实中番茄红素测定方法的研究：
本研究中高效液相色谱法(HPLC)以甲醇-乙腈-二氯甲烷(20：75：5)为流动相,502nm为检测波长,各参数符合分析要求,实验结果可靠,该法能较好的对西瓜果实中的番茄红索做定性定量分析；以2%二氯甲烷石油醚为提取液,502nm波长为测定吸光值的分光光度计法检测西瓜番茄红素含量平均约为HPLC法的1.45倍；分光光度计法测定结果与HPLC去具有一致性,可用于不同品种之间番茄红素含量的比较。
2.不同倍性西瓜果实中番茄红素含量的测定：
2.1不同倍性西瓜果实发育过程中番茄红素含量的变化：红瓤西瓜随果实发育番茄红素的含量不断增多；露地种植的L1、L2,L3和L4四组不同倍性材料之间,二倍体中的番茄红索前期积累较快,三倍体和四倍体前期积累相对较慢但是后期能迅速积累,并且超过二倍体中番茄红素的含量；春季大棚种植的PLl和PL3两个组合,多倍体西瓜果实的番茄红素积累速度和二倍体一致,甚至多倍体要稍快于二倍体。
2.2不同倍性西瓜果实授粉后30天番茄红素含量的差别：从平均值来看,不同倍性西瓜果实中番茄红素含量是三倍体>四倍体>二倍体,二倍体和多倍体在P=0.05水平上存在显著差异,三倍体和四倍体之间不存在显著性差异。
3.不同倍性西瓜果肉细胞中有色体的变化：
不同倍性西瓜细胞中有色体以球状、针状、环状、聚集状等多种结构存在；随着果实的发育,有色体结构不断变化,数目从少到多,形状从非对称到完全对称,最后在过熟西瓜的果肉细胞中以无组织、不对称结构存在；二倍体西瓜中有很多聚集状番茄红素结晶体,也有球状、针状,环状结构的有色体,三倍体西瓜中有色体以球状、针状、环状等多种结构大量存在,四倍体西瓜中球状、聚集状等结构的有色体较多,并且有色体体积相对较大。
4.不同倍性西瓜果实中番茄红素关键酶基因表达的定量分析：
4.1西瓜果实发育过程中番茄红素关键酶基因表达的定量分析：L1和L2两个组合随着果实发育时期的变化,PSY-A和LCYB两个基因的表达量均是先增高后降低：不同倍性之间,基因的表达量存在着明显的差异,多倍体中两个基因的表达量要比二倍体高。
4.2四倍体西瓜品种PSY-A和LCYB基因的时空表达分析：PSY-A和LCYB基因在西瓜不同组织器官中均有表达,而且具有明显差异；不同组织间比较,PSY-A和LCYB基因在雄花中的表达量均最高,根中最低；果实发育过程中,两个基因都是先升高后降低,后期LCYB急剧降低,此时番茄红素也大量合成并积累。
4.3番茄红素积累与酶基因表达的关系：不同倍性西瓜果实中番茄红素含量均随着果实的发育而不断增加,前期,PSY-A和LCYB基因的表达量逐渐增高,番茄红素逐渐积累,后期PSY-A和LCYB基因的表达的表达量均降低,特别是LCYB基闪的表达量急剧下降,番茄红索此时大量积累。
In this study, the contents of lycopene during watermelon fruits development with different cultivar and different ploidy including diploid (2X), triploid (3X) and tetraploid (4X) were measured, the gene expression levels of phytoene synthase (PSY-A) and lycopene b-cyclase (LCYB) were determined. The cell and subcelluar structure, especially plastids were studied by transmission electron microscopy TEM). The results would provide a theoretical basis for optimization of high-lycopene watermelon breeding. The main results were as follows:
1. The comparison of two determination methods of lycopene contents in watermelon:
The contents of lycopene in different ploidy watermelon fruits determined by high perfonnance liquid chromatography(HPLC) and spectrophotometric method were compared, a more accurate and convenient method was investigated. The result showed that:The HPLC method was simple and accurate with the mobile phase methanol:acetonitrile:dichloromethane(20:75:5) detected at502nm.The determination data of the purity of lycopene by UV(Ultraviolet Visible Spectrophotometer) were1.45multiples of the data by HPLC. The two sets datas for different methods were consistency.
2. The changes of lycopene content in watermelon fruits
2.1The lycopene content was increasing during watermelon fruit development(10to30days after pollination, DAP). Riper fruits had higher lycopene concent than unmature fruits. Four lines (L1、L2, L3and L4) showed that diploid watermelon developed fast and accumulated more lycopene in the early stage, the lycopene contents of triploid and tetraploid watermelon increased most form20DAP after pollination, lycopene increased faster and accumulated much more than diploid30DAP; But lines of PL1and PL3showed that the polyploidy watermelon developed quickly and accumulated more lycopene.
2.2The lycopene content in different ploidy watermelon was triploid> tetraploid> diploid at the average level; The diploid and ployploid have significant differences at the level of P=0.05, there is no significant differences between triploid and tetraploid.
3. The cell and subcelluar structure, especially plasmids were compaired by transmission electron microscopy. It was found that the plastids structure changing with the fruit development, from less to more,from asymmetric to completely symmetrical and finally unorganized in over matured fruit; chromoplasts had many forms in watermelon fruit including globular structure, ring-like structure, thread-like structure and other structures; Some carotenoids congeries together.
4. The transcriptional expression difference of PSY-A and LCYB gene during fruits development with different cultivar:
4.1The expression of PSY-A and LCYB gene participated in lycopene synthetic were quantitative by real-time quantitative PCR. During fruit maturation, the expression levels of PSY-A and LCYB increased during the mid-stages but decreased in the fully mature fruit; the expression levels of PSY-A and LCYB were significant differences in L1and L2, the two gene expression of triploid were lowest in Llbut highest in L2.
4.2The PSY-A and LCYB gene were expression in different organs of tetraploid watermelon, but the expression level had differences, they are high in the male flowers match to the color; The highest transcript levels were detected around the large green-red stages (25DAP), a period when carotenoid synthesis is probably greatest.Then The dramatic reduction in expression of LCYB may be a result of the massive accumulation of lycopene in the ripening fruit.
4.3The relationship between lycopene accumulation and gene expression. During fruit maturation, the expression levels of PSY-A and LCYB increased during the mid-stages but decreased in the fully mature fruit. In addition, lycopene accumulated as the flesh changed from green to red. Thus, PSY-A and LCYB expression correlated with lycopene accumulation during fruit maturation. Riper fruits had higher lycopene concentrations than less ripe fruits.
1.西瓜果实中番茄红素测定方法的研究：
本研究中高效液相色谱法(HPLC)以甲醇-乙腈-二氯甲烷(20：75：5)为流动相,502nm为检测波长,各参数符合分析要求,实验结果可靠,该法能较好的对西瓜果实中的番茄红索做定性定量分析；以2%二氯甲烷石油醚为提取液,502nm波长为测定吸光值的分光光度计法检测西瓜番茄红素含量平均约为HPLC法的1.45倍；分光光度计法测定结果与HPLC去具有一致性,可用于不同品种之间番茄红素含量的比较。
2.不同倍性西瓜果实中番茄红素含量的测定：
2.1不同倍性西瓜果实发育过程中番茄红素含量的变化：红瓤西瓜随果实发育番茄红素的含量不断增多；露地种植的L1、L2,L3和L4四组不同倍性材料之间,二倍体中的番茄红索前期积累较快,三倍体和四倍体前期积累相对较慢但是后期能迅速积累,并且超过二倍体中番茄红素的含量；春季大棚种植的PLl和PL3两个组合,多倍体西瓜果实的番茄红素积累速度和二倍体一致,甚至多倍体要稍快于二倍体。
2.2不同倍性西瓜果实授粉后30天番茄红素含量的差别：从平均值来看,不同倍性西瓜果实中番茄红素含量是三倍体>四倍体>二倍体,二倍体和多倍体在P=0.05水平上存在显著差异,三倍体和四倍体之间不存在显著性差异。
3.不同倍性西瓜果肉细胞中有色体的变化：
不同倍性西瓜细胞中有色体以球状、针状、环状、聚集状等多种结构存在；随着果实的发育,有色体结构不断变化,数目从少到多,形状从非对称到完全对称,最后在过熟西瓜的果肉细胞中以无组织、不对称结构存在；二倍体西瓜中有很多聚集状番茄红素结晶体,也有球状、针状,环状结构的有色体,三倍体西瓜中有色体以球状、针状、环状等多种结构大量存在,四倍体西瓜中球状、聚集状等结构的有色体较多,并且有色体体积相对较大。
4.不同倍性西瓜果实中番茄红素关键酶基因表达的定量分析：
4.1西瓜果实发育过程中番茄红素关键酶基因表达的定量分析：L1和L2两个组合随着果实发育时期的变化,PSY-A和LCYB两个基因的表达量均是先增高后降低：不同倍性之间,基因的表达量存在着明显的差异,多倍体中两个基因的表达量要比二倍体高。
4.2四倍体西瓜品种PSY-A和LCYB基因的时空表达分析：PSY-A和LCYB基因在西瓜不同组织器官中均有表达,而且具有明显差异；不同组织间比较,PSY-A和LCYB基因在雄花中的表达量均最高,根中最低；果实发育过程中,两个基因都是先升高后降低,后期LCYB急剧降低,此时番茄红素也大量合成并积累。
4.3番茄红素积累与酶基因表达的关系：不同倍性西瓜果实中番茄红素含量均随着果实的发育而不断增加,前期,PSY-A和LCYB基因的表达量逐渐增高,番茄红素逐渐积累,后期PSY-A和LCYB基因的表达的表达量均降低,特别是LCYB基闪的表达量急剧下降,番茄红索此时大量积累。
In this study, the contents of lycopene during watermelon fruits development with different cultivar and different ploidy including diploid (2X), triploid (3X) and tetraploid (4X) were measured, the gene expression levels of phytoene synthase (PSY-A) and lycopene b-cyclase (LCYB) were determined. The cell and subcelluar structure, especially plastids were studied by transmission electron microscopy TEM). The results would provide a theoretical basis for optimization of high-lycopene watermelon breeding. The main results were as follows:
1. The comparison of two determination methods of lycopene contents in watermelon:
The contents of lycopene in different ploidy watermelon fruits determined by high perfonnance liquid chromatography(HPLC) and spectrophotometric method were compared, a more accurate and convenient method was investigated. The result showed that:The HPLC method was simple and accurate with the mobile phase methanol:acetonitrile:dichloromethane(20:75:5) detected at502nm.The determination data of the purity of lycopene by UV(Ultraviolet Visible Spectrophotometer) were1.45multiples of the data by HPLC. The two sets datas for different methods were consistency.
2. The changes of lycopene content in watermelon fruits
2.1The lycopene content was increasing during watermelon fruit development(10to30days after pollination, DAP). Riper fruits had higher lycopene concent than unmature fruits. Four lines (L1、L2, L3and L4) showed that diploid watermelon developed fast and accumulated more lycopene in the early stage, the lycopene contents of triploid and tetraploid watermelon increased most form20DAP after pollination, lycopene increased faster and accumulated much more than diploid30DAP; But lines of PL1and PL3showed that the polyploidy watermelon developed quickly and accumulated more lycopene.
2.2The lycopene content in different ploidy watermelon was triploid> tetraploid> diploid at the average level; The diploid and ployploid have significant differences at the level of P=0.05, there is no significant differences between triploid and tetraploid.
3. The cell and subcelluar structure, especially plasmids were compaired by transmission electron microscopy. It was found that the plastids structure changing with the fruit development, from less to more,from asymmetric to completely symmetrical and finally unorganized in over matured fruit; chromoplasts had many forms in watermelon fruit including globular structure, ring-like structure, thread-like structure and other structures; Some carotenoids congeries together.
4. The transcriptional expression difference of PSY-A and LCYB gene during fruits development with different cultivar:
4.1The expression of PSY-A and LCYB gene participated in lycopene synthetic were quantitative by real-time quantitative PCR. During fruit maturation, the expression levels of PSY-A and LCYB increased during the mid-stages but decreased in the fully mature fruit; the expression levels of PSY-A and LCYB were significant differences in L1and L2, the two gene expression of triploid were lowest in Llbut highest in L2.
4.2The PSY-A and LCYB gene were expression in different organs of tetraploid watermelon, but the expression level had differences, they are high in the male flowers match to the color; The highest transcript levels were detected around the large green-red stages (25DAP), a period when carotenoid synthesis is probably greatest.Then The dramatic reduction in expression of LCYB may be a result of the massive accumulation of lycopene in the ripening fruit.
4.3The relationship between lycopene accumulation and gene expression. During fruit maturation, the expression levels of PSY-A and LCYB increased during the mid-stages but decreased in the fully mature fruit. In addition, lycopene accumulated as the flesh changed from green to red. Thus, PSY-A and LCYB expression correlated with lycopene accumulation during fruit maturation. Riper fruits had higher lycopene concentrations than less ripe fruits.
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