拟南芥atcwinv1基因T-DNA插入纯合突变体PCR鉴定及表型观察阮燕晔;张莹;王波【摘要】以拟南芥atcwinvl 基因T-DNA插入纯合突变体和野生型植株为材料,比较研究了2种基因型植株在营养期和生殖期的形态差异.结果表明:拟南芥atcwinvl 基因T-DNA插入纯合突变体(简称突变体)较野生型萌发率平均下降5.88个百分点;突变体在44 d抽薹,较野生型延后4d;分支数平均4支,较野生型下降20.84%;果荚开裂时间6d左右,较野生型延长2d;单株果荚数平均62.27个,较野生型降低11.00%;单株果荚种粒数平均45.87粒,较野生型降低21.46%;突变体的单果荚长度平均14.52 cm,较野生型降低10.24%;单株果质量平均50.83mg,较野生型降低23.70%.拟南芥突变体在营养生长时期的株高平均10.44 cm,较野生型下降21.03%;主根长平均7.62 cm,较野生型下降14.96%;单株莲座叶面积平均3.16 cm2,较野生型下降13.90%;单株地上部分鲜质量平均81.81mg,较野生型下降11.11%;单株根鲜质量平均6.21mg,较野生型下降17.64%;单株地上部分干质量平均6.17 mg,较野生型下降15.60%;单株根干质量平均0.55mg,较野生型下降6.78%.拟南芥突变体在生殖生长时期的株高平均18.78 cm,较野生型增加4.22%;主根长平均16.48 cm,较野生型下降5.88%;单株莲座叶面积平均6.80 cm2,较野生型下降6.21%;单株地上部分鲜质量平均129.85 mg,较野生型下降9.69%;单株根鲜质量平均9.97 mg,较野生型下降13.23%;单株地上部分干质量平均9.22 mg,较野生型下降4.16%;单株根干质量平均0.70mg,较野生型下降6.67%.以上研究结果表明,atcwinvl 基因T-DNA插入突变影响了拟南芥植株正常的生长发育.%In the research, homozygous mutant (atcwinvl) and wild type of Arabidopsis were used to compare plant morphological differences in the period ofvegetative growth and reproductive growth. The results showed that compared to the wild type the germination rate of atcwinvl averagely decreased by 5.88 percentage points; compared to the wild type, bolting time of atcwinvl was 44 d, delayed 4 days;branch number of atcwinvl was 4, decreased by 20. 84 ％ ;cracking time of the pod of atcwinvl was 6,delayed 2 days;fruit pod number per plant of atcwinvl was 62.27,decreased by11.00％ ,fruit number of a pod of atcwinvl was 45.87,decreased by21.46％;length of a fruit pod of atcwinvl was 14.52cm,decreased by 10. 24％fruit weight of a plant of atcwinvl was 50. 83mg,decreased by 23.70％. In the period of vegetative growth,compared to the wild type, plant height of atcwinvl was 10.44cm, decreased by 21. 03％; root length of atcwinvl was 7.62cm, average decreased by 14. 96％; area of rosette leaf of atcwinvl was 3. 16 cm2 ,decreased by 13.90％ ;aboveground fresh weight of per plant was 81.81 mg, decreased by 11.11％ ;fresh weight of root per plant was6.21mg, decreased by 17.64％ ;aboveground dry weight of per plant was 6.17 mg, decreased by 15.60 ％ ;dry weight of root per plant was 0. 55mg,decreased by 6. 78 ％ ;In the period of reproductive growth, compared to the wild type, plant height of atcwinvl was 18. 78cm,increased by4.22％ ;root length of atcwinvl was 16.48cm,averagely decreased by5.88％; area of rosette leaf of atcwinvl was6.80 cm2, decreased by 6.21％; fresh weight of a plant of atcwinvl was 129.85 mg,decreased by 9.69％ ;fresh weight of root per plant was 9. 97 mg,decreased by 13.23％ ;aboveground dry weight of per plant was 9.22 mg, decreased by 4. 16％ ;dry weight of root per plant was 0. 70mg,decreased by 6.67％. The results obtainedshowed that growth of Arabidopsis was influenced by T-DNA insertional mutagenesis of atcwinvl gene.【期刊名称】《河南农业科学》【年(卷),期】2011(040)005【总页数】5页(P62-66)【关键词】拟南芥;atcwinvl基因;T-DNA插入突变;基因功能【作者】阮燕晔;张莹;王波【作者单位】沈阳农业大学,生物科学技术学院,辽宁沈阳110866;沈阳农业大学,生物科学技术学院,辽宁沈阳110866;沈阳农业大学,生物科学技术学院,辽宁沈阳110866【正文语种】中文【中图分类】Q789蔗糖是高等植物体内光合产物运输与分配的主要形式,其在植株中定向运输和分配的方式不仅调控植株的整个生长和发育进程,也决定作物的产量和品质。

而在蔗糖的库器官卸载过程中,细胞壁转化酶发挥着重要的作用[1-2]。

目前,对细胞壁转化酶的研究主要集中在其基因克隆和表达方面。

细胞壁转化酶基因最先从胡萝卜中分离得到[3],随后在马铃薯、拟南芥、烟草、番茄和葡萄等植物中也分离到编码该酶的全长或部分的核苷酸序列。

细胞壁转化酶基因在质外体中表达最活跃,并通过增加蔗糖的浓度梯度加快蔗糖卸载[4]。

此外,细胞壁转化酶还在蔗糖水解[4]、渗透调节[5]和伤害胁迫[3,6]中起作用。

目前,在模式植物拟南芥中分离出了4个编码细胞壁转化酶的相关基因,分别是atcwinv1,atcwinv2,atcwinv4,atcwinv5,其中 atcwinv1基因的表达水平最高,被普遍认为是蔗糖在韧皮部卸载的一个主效基因[7],但其确切的生理生化功能和作用机制还不十分清楚。

目前,拟南芥基因组测序已经完成,而且有大量的T -DNA 插入突变体,从中可以获得细胞壁转化酶基因敲出的突变体[8],因而是用反向遗传学方法研究植物细胞壁转化酶基因的最理想的材料。

通过比较拟南芥细胞壁转化酶基因纯合突变体与野生型拟南芥生长发育以及生理特征,可以获得拟南芥细胞壁转化酶基因的生理功能的相关信息。

鉴此,对拟南芥atcwinv1基因 TDNA插入纯合突变体进行表型观察,揭示该基因缺失对拟南芥植株生长发育的影响,为进一步研究该基因生理功能奠定理论基础。

1 材料和方法1.1 材料atcwinv1基因T -DNA 插入拟南芥种子（编号为salk_091455,原种为Columbia-0型）由拟南芥生物资源中心提供,拟南芥野生型（Col-0）种子由英国伯明翰大学提供。

1.2 方法1.2.1 拟南芥培养拟南芥atcwinv1型和野生型种子经4℃下春化3d后播种于盆土（泥炭土∶蛭石=3∶1）。

2种基因型种子分3批培养,每批分别播种100盆,每盆栽种1株拟南芥。

将种好的拟南芥置于长日照周期为10h光照/14h黑暗、光照强度为150μ mol/（m2◦s）、相对湿度为 70%白天/80%夜间和温度为（22±1）℃的培养室内生长[9]。

每批植株生长到35d时（营养期）收获1次,每次收获50株;55d时（生殖期）收获1次,每次收获50株,收获植株全部用于表型性状观测。

1.2.2 atcwinv1纯合突变体PCR鉴定采用三引物PCR法,以所提取的拟南芥植株总 DNA为模板,进行 PCR扩增[10-11]。

atcwinv1基因引物由宝生物工程有限公司合成,3条引物分别为LP:5′-TCT TCCCTATATT TGCAAGCG-3′;RP:5′-TGGT TTCAAGATGGACGGTAC-3′;LBa1:5′-ATT TTGCCGAT TTCGGAAC-3′。

反应体系共20μ L:1μ L 模板、1μ L（20μ mol/L）引物、0.4μ L（10mmol/L）dNTPs、2μ L（10 ×PCR）缓冲液、3.6μ L（25mmol/L）MgCl2、Taq DNA 聚合酶 1U （BBI）、双蒸水1μ L,甘油10μ L。

循环参数（30次循环）:94℃,1min;58℃,1min;72℃,2min。

选用1.7%的琼脂糖凝胶（内含0.5mg/L Goldveiw）电泳检测后,用凝胶成像系统在300nm波长的透射光下照相并记录扩增结果。