木薯SBEⅡ和MeEFⅠ基因物理定位的研究
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摘要
木薯(Manihot esculenta Crantz)是世界三大薯类作物之一,同时也是重要的热带能源植物。围绕着木薯高产优质、抗逆性等相关性状,前人开展了一系列分子生物学研究,已从木薯中克隆了多种重要基因或cDNA。例如,颗粒结合型淀粉合成酶基困(gbss)、淀粉分支酶基因(she)、α-羟腈酶基因(hnl)、甘油醛-3-磷酸脱氢酶基因(G3Pdh)、苯丙氨酸解氨酶基因(PAL2)、延长因子基因(MeEF1)、富含羟脯氨酸糖蛋白基因(HRGP)、铜锌超氧化物歧化酶基因(sod)等。然而,这些基因在木薯染色体上的位置和分布特点等至今尚未见报道。本研究通过原位PCR技术对木薯淀粉分支酶相关基因(SBEⅡ)和木薯延长因子(MeEFⅠ)进行了基因定位,结果如下
设计、合成及筛选了2对特异扩增木薯淀粉分支酶基因(SBEⅡ)和木薯延长因子(MeEFⅠ)的引物。
在原先甘庶、橡胶树原位PCR技术体系的基础上,确立了适合于木薯的原位PCR技术体系。该原位PCR反应液体积为50μl,各反应组分的终浓度为：1×Buffer, 4.5 mM MCl2,0.5 mg/ml BSA,200μM的dATP、dGTP、dGTP,72μm dTTP,4μm DIG-Ⅱ-dUTP,5 U/5μ1的Taq DNA聚合酶,2μM的引物。扩增程序为：PCR扩增,先95℃变性10min,然后94℃变性1min,55℃退火1 min(视引物的Tm值),72℃延仲1.5 min,共20个循环,最后72℃延伸10min。操作流程为：5 ug/ml胃蛋白酶5 min→0.5×TBS 10min→灭菌水2×5 min→70%甲酰胺70℃变性2min→0.1×SSC冰浴1min→灭菌水冰浴1min→乙醇脱水→PCR扩增。扩增后处理流程为：0.1×PBS 37℃4-5 min→5% BSA 37℃孵育20 min→20μg/ml Anti-DIG-Fluorescein37℃孵育1 h→0.1×SSC/吐温20漂洗2×4 min→1μg/μl PI37℃孵育15 min→0.1×SSC/吐温20漂洗2×1 min→抗褪色剂Vectashield封片检测。
用SBEⅡ特异引物在华南6号的根尖(2n=36)中期细胞的染色体标本进行原位PCR扩增,在不同分裂时期的细胞核中均能发现1-2个信号位点,初步将木薯特异DNA序列SBEⅡ丛因定位于木薯华南6号的第7号染色体的长臂上,扩增位点到着丝粒的百分距离是63.80。
用MeEFⅠ特异引物在华南6号的根尖(2n=36)中期细胞的染色体标本进行原位PCR扩增,在不同分裂时期的细胞核中均能发现1-2个信号位点,将木薯特异DNA序列MeEFⅠ基因定位于木薯华南6号的第10号染色体的短臂上,扩增位点到着丝粒的百分距离是35.11。,
对木薯华南6号进行了核型分析,华南6号的染色体数目为2n=36,染色体相对长度为3.66%-7.35%,笫2、7、18为近中着丝粒染色体(sm),其余的为中着丝粒染色体(m)。最长与最短染色体的长度比为2,平均臂比为1.44,第2、9号染色体为随体染色体。核型公式为2n=36=30m(1 sat)+6sm(1 sat)。按照Sttebbins的方法,核型分类属于2B型。
Cassava is one of the three potato crops in world, and is an important economic crop. Many important genes or cDNAs about high yield and steessing resistance have been cloned, for example granule-bound starch synthase (gbss)、starch branching enzyme (sbe)、manihot esculenta elongation factor 1-alpha (MeEF1) Alpha-hydroxynitrile lyase (hnl)、glyceraldehyde 3-phosphate dehydrogenase (G3pdh)、Phenylalanine ammonia-lyase (PAL)、hydroxyproline-rich glycoprotein (HRGP)、copper/zinc superoxide dismutase (SOD) etc, but few of them has been reported physically located onto chromosomes. Starch branching enzymeⅡ(SBEⅡ) and manihot esculenta elongation factor 1-alpha (MeEFl) gene were physically located onto chromosomes in SC6 by in situ PCR . The results were as felloeing:
Get two primers which is specific to Starch branching enzyme (SBE) and manihot esculenta elongation factor 1-alpha (MeEF1).
The method of direction in situ PCR on chromosomes of cassava was discussed and a complete set of method was found in this study by refered the methods in Hevea and sugarcane. The total volume of direction in situ PCR reaction was 50μl, which contained 1×Buffer, 4.5 mM MCl2, 0.5 mg/ml BSA, 5 U/5μl Taq DNA Polynerase, 200uM dATP, dGTP and dGTP, 72 uM dTTP, 4μM DIG-Ⅱ-dUTP, 2μM primers. The in stu PCR amplification procedure was like this: Predenaturalization 10 min with 95℃, then 20 cycles including denaturalization of 94℃for 1 min, anneling at 55°C (by Tin of primer) for 1 min, extension at 72℃for 1.5 min, the last stop was extenstion at. 72°C for 10 min. The operational process of in situ PCR was 5 ug/ml pepsin 5 min→0.5×TBS 10min→sterilized water 2×5 min→70% deionized formamide 70℃denatureed 2min→0.1×SSC ice-bath 1min→sterilized water ice-bathlmin→ethanol dehydrated→PCR amplification. The process of after PCR amplification was: 0.1×PBS 37℃4-5 min→5% BSA 37℃20 min→20μg/ml Anti-DIG-Fluorescein 37℃1 h→0.1×SSC/Tween 20 washed 2×4 min→1μg/μl PI 37℃15 min→0.1×SSC/ Tween 20 washed 2×1 min→coverslipping with Vectashield→signal detected..
The signals were detected on the different phases of the cells of SC6 roots tip (2n=36) used the developed in situ PCR method. The signal site of the SBEⅡgene distributed on the long arm of the seventh chromosomes on SC6, the pereent distances from centromere to detection site was 63.80.
The sign site of the MeEFl gene distributed on the short arm of the seventh chromosomes on SC6, the pereent distanees from centromere to detection site was 35.11.
Karyotypes on SC6 were analysed. The number of chromosomes on SC6 was 36, the relative length was 3.66%-7.35%, the second、seventh and the Eighteenth chromosomes was sub-median kinetochores, others were median kinetochores, the rato between longest and shortest chromosome was 2,the average arm ratio was 1.44, the karyotype formula of SC6 was 2n=36=33m (2sat)+3sm (1sat), the karyotype belonged to 2B type.
设计、合成及筛选了2对特异扩增木薯淀粉分支酶基因(SBEⅡ)和木薯延长因子(MeEFⅠ)的引物。
在原先甘庶、橡胶树原位PCR技术体系的基础上,确立了适合于木薯的原位PCR技术体系。该原位PCR反应液体积为50μl,各反应组分的终浓度为：1×Buffer, 4.5 mM MCl2,0.5 mg/ml BSA,200μM的dATP、dGTP、dGTP,72μm dTTP,4μm DIG-Ⅱ-dUTP,5 U/5μ1的Taq DNA聚合酶,2μM的引物。扩增程序为：PCR扩增,先95℃变性10min,然后94℃变性1min,55℃退火1 min(视引物的Tm值),72℃延仲1.5 min,共20个循环,最后72℃延伸10min。操作流程为：5 ug/ml胃蛋白酶5 min→0.5×TBS 10min→灭菌水2×5 min→70%甲酰胺70℃变性2min→0.1×SSC冰浴1min→灭菌水冰浴1min→乙醇脱水→PCR扩增。扩增后处理流程为：0.1×PBS 37℃4-5 min→5% BSA 37℃孵育20 min→20μg/ml Anti-DIG-Fluorescein37℃孵育1 h→0.1×SSC/吐温20漂洗2×4 min→1μg/μl PI37℃孵育15 min→0.1×SSC/吐温20漂洗2×1 min→抗褪色剂Vectashield封片检测。
用SBEⅡ特异引物在华南6号的根尖(2n=36)中期细胞的染色体标本进行原位PCR扩增,在不同分裂时期的细胞核中均能发现1-2个信号位点,初步将木薯特异DNA序列SBEⅡ丛因定位于木薯华南6号的第7号染色体的长臂上,扩增位点到着丝粒的百分距离是63.80。
用MeEFⅠ特异引物在华南6号的根尖(2n=36)中期细胞的染色体标本进行原位PCR扩增,在不同分裂时期的细胞核中均能发现1-2个信号位点,将木薯特异DNA序列MeEFⅠ基因定位于木薯华南6号的第10号染色体的短臂上,扩增位点到着丝粒的百分距离是35.11。,
对木薯华南6号进行了核型分析,华南6号的染色体数目为2n=36,染色体相对长度为3.66%-7.35%,笫2、7、18为近中着丝粒染色体(sm),其余的为中着丝粒染色体(m)。最长与最短染色体的长度比为2,平均臂比为1.44,第2、9号染色体为随体染色体。核型公式为2n=36=30m(1 sat)+6sm(1 sat)。按照Sttebbins的方法,核型分类属于2B型。
Cassava is one of the three potato crops in world, and is an important economic crop. Many important genes or cDNAs about high yield and steessing resistance have been cloned, for example granule-bound starch synthase (gbss)、starch branching enzyme (sbe)、manihot esculenta elongation factor 1-alpha (MeEF1) Alpha-hydroxynitrile lyase (hnl)、glyceraldehyde 3-phosphate dehydrogenase (G3pdh)、Phenylalanine ammonia-lyase (PAL)、hydroxyproline-rich glycoprotein (HRGP)、copper/zinc superoxide dismutase (SOD) etc, but few of them has been reported physically located onto chromosomes. Starch branching enzymeⅡ(SBEⅡ) and manihot esculenta elongation factor 1-alpha (MeEFl) gene were physically located onto chromosomes in SC6 by in situ PCR . The results were as felloeing:
Get two primers which is specific to Starch branching enzyme (SBE) and manihot esculenta elongation factor 1-alpha (MeEF1).
The method of direction in situ PCR on chromosomes of cassava was discussed and a complete set of method was found in this study by refered the methods in Hevea and sugarcane. The total volume of direction in situ PCR reaction was 50μl, which contained 1×Buffer, 4.5 mM MCl2, 0.5 mg/ml BSA, 5 U/5μl Taq DNA Polynerase, 200uM dATP, dGTP and dGTP, 72 uM dTTP, 4μM DIG-Ⅱ-dUTP, 2μM primers. The in stu PCR amplification procedure was like this: Predenaturalization 10 min with 95℃, then 20 cycles including denaturalization of 94℃for 1 min, anneling at 55°C (by Tin of primer) for 1 min, extension at 72℃for 1.5 min, the last stop was extenstion at. 72°C for 10 min. The operational process of in situ PCR was 5 ug/ml pepsin 5 min→0.5×TBS 10min→sterilized water 2×5 min→70% deionized formamide 70℃denatureed 2min→0.1×SSC ice-bath 1min→sterilized water ice-bathlmin→ethanol dehydrated→PCR amplification. The process of after PCR amplification was: 0.1×PBS 37℃4-5 min→5% BSA 37℃20 min→20μg/ml Anti-DIG-Fluorescein 37℃1 h→0.1×SSC/Tween 20 washed 2×4 min→1μg/μl PI 37℃15 min→0.1×SSC/ Tween 20 washed 2×1 min→coverslipping with Vectashield→signal detected..
The signals were detected on the different phases of the cells of SC6 roots tip (2n=36) used the developed in situ PCR method. The signal site of the SBEⅡgene distributed on the long arm of the seventh chromosomes on SC6, the pereent distances from centromere to detection site was 63.80.
The sign site of the MeEFl gene distributed on the short arm of the seventh chromosomes on SC6, the pereent distanees from centromere to detection site was 35.11.
Karyotypes on SC6 were analysed. The number of chromosomes on SC6 was 36, the relative length was 3.66%-7.35%, the second、seventh and the Eighteenth chromosomes was sub-median kinetochores, others were median kinetochores, the rato between longest and shortest chromosome was 2,the average arm ratio was 1.44, the karyotype formula of SC6 was 2n=36=33m (2sat)+3sm (1sat), the karyotype belonged to 2B type.
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