海岛棉蔗糖合成酶基因克隆及生物信息学和表达模式分析

Cloning between Bioinformatics and Expression Pattern Analysis of Sucrose Synthase in Island Cotton(Gossypium barbadense L.)

蔗糖合成酶(Sucrose Synthase,EC 2.4.1.13,Sus)在植物不同组织及不同发育阶段起不同的作用,是参与植物糖类代谢调控的关键酶之一。本研究,我们利用目前已完全确定的亚洲棉(Gossypium arboretum L.)Sus基因7个成员(Ga Sus1-Ga Sus7,Gene Bank登录号JQ995522-JQ995528)进行同源克隆并通过q RT-PCR方法对不同纤维发育时期Sus表达模式进行了检测。得到的两个组配海岛棉(Gossypium barbadense L.)重组自交系(recombinant inbred line,RIL,F2:6)亲本材料5917(高比强度品种,平均比强度48.2 c N.tex-1)和Pima-7(低比强度品种,平均比强度37.6 c N.tex-1)的Sus g DNA序列。利用生物信息学方法对基因结构特征分析比较发现,5917、Pima-7与亚洲棉Sus编码区(CDS)中分别存在(Sus1与Ga Sus1:9,8;Sus2与Ga Sus2:25,8;Sus3与Ga Sus3:8,9;Sus4与Ga Sus4:40,41;Sus5与Ga Sus5:27,27;Sus6与Ga Sus6:16,16;Sus7与Ga Sus7:7,7)处单核苷酸多态性(SNP)位点。两个海岛棉品种间Sus1,Sus2,Sus3,Sus4 CDS中存在(1,21,4,1)处差异SNP位点,Sus5,Sus6,Sus7 CDS中无差异。海岛棉Sus CDS中核酸序列碱基置换虽改变了相应氨基酸组成,但氨基酸理化性质和蛋白质结构与功能均与亚洲棉相同未发生明显变化。海岛棉纤维发育过程中,Sus在纤维中的表达具有特异性。本研究在设立高、低比强度研究对象并比对8个纤维生育时期的基础上进一步发现Sus3在纤维次生壁加厚初期(20 DPA)开始大量表达到达峰值随后下降。高比强度海岛棉品种5917 Sus1除在纤维发育起始期(0 DPA^5 DPA)大量表达外,在纤维次生壁加厚后期和末期(30 DPA^35 DPA)表达量又重新升高,低比强度海岛棉品种Pima-7 Sus1无此回升现象且表达量远低于前者。研究表明,Sus3在纤维次生壁加厚初期起作用,Sus1作用于纤维次生壁加厚后期和末期,这两个Sus在纤维次生壁加厚期发挥重要的作用,可能与纤维比强度的形成和差异有关。

As one of key enzymes involved in the regulation of carbohydrate metabolism. Sucrose synthase plays a vital role to different plant tissues and in different development stages. In this study, we used Sus gene that had been fully established from asian cotton(Gossypium arboretum L.)(Ga Sus1-Ga Sus7, Gene Bank Accession No:JQ995522-JQ995528) to carry out homology cloning. In addition, the expression pattern of Sus 7 members during different fiber development periods was detected by q RT-PCR. Sequences of Sus g DNA had been Obtained from5917(variety of high fiber strength, the average strength: 48.2 c N. tex^-1) with Pima-7(variety of low fiber strength,the average strength: 37.6 c N. tex^-1), which were two parent materials of recombinant inbred lines(RIL F-_(2:6)) from island cotton(Gossypium barbadense L.). When analysis and comparison structure characteristic of gene coding region(CDS) among 5917, Pima-7 and Gossypium arboretum L. through bioinformatics method, we could discovery that exist single nucleotide polymorphism sex site(SNP) in there, repectively(Sus1 with Ga Sus1: 9, 8; Sus2 with Ga Sus2: 25, 8; Sus3 with Ga Sus3: 8, 9; Sus4 with Ga Sus4: 40, 41; Sus5 with Ga Sus5: 27, 27; Sus6 with Ga Sus6: 16, 16; Sus7 with Ga Sus7: 7, 7). When compare 5917 and Pima-7. Sus1, Sus2, Sus3, Sus4 were exist different SNP site in CDS(1, 21, 4, 1), while no difference existed in the CDS of Sus5, Sus6, Sus7 gene. Although,the composition of amino acid from Sus gene CDS was changed due to the base replacement of nucleotide sequence, the physical and chemical properties of amino acid as well as structures and functions of protein remained unchanged, which were roughly the same as the Gossypium arboretum L. The expression of Sus gene shows specific characteristics during fiber development periods. In our study, based on setting up objects of higher and lower fiber strength and comparison of 8 fiber growth periods respectively. It further found that when began to massively express raised to the peak and go down followed of expression in Sus3 during early of secondary wall thickening(20 DPA). To abundantly express in Sus1 at initial period(0 DPA^5 DPA) during fiber development,while it was increased again at anaphase and final periods during secondary wall thickening(30 DPA ~35 DPA)from 5917. For Pima-7, it did not exist rerise phenomenon of expression and much less than the former. Research shows that, Sus3 mainly takes part in early of secondary wall thickening, while Sus1 mainly works in anaphase and final of secondary wall thickening. Both of them play an important role in secondary wall thickening, which is mainly relate to the formation and differences of fiber strength.