菠萝八氢番茄红素脱氢酶基因Aco-PDS1编辑株系的构建  

Phytoene Desaturase Gene Aco-PDS1 was Edited in Pineapple Genome

作　　者：舒海燕[1] 王悠 冼淑云 李科明[1] 詹儒林 常胜合[1] Shu Haiyan;Wang You;Xian Shuyun;Li Keming;Zhan Rulin;Chang Shenghe(Haikou Experimental Station,Chinese Academy of Tropical Agricultural Sciences,Haikou,571101)

机构地区：[1]中国热带农业科学院海口实验站,海口571101

出　　处：《分子植物育种》2022年第22期7446-7452,共7页Molecular Plant Breeding

基　　金：国家重点研发计划项目(2019YFD1001104)资助。

摘　　要：为建立菠萝基因编辑技术体系,本研究对菠萝八氢番茄红素脱氢酶基因进行了分析,发现菠萝基因组中有两个八氢番茄红素脱氢酶基因,其中Aco-PDS1与其他植物PDS序列相似度高于Aco-PDS2与其他植物PDS序列的相似度。在根、叶、冠芽、未成熟果肉、成熟果肉中,Aco-PDS1的表达量均高于Aco-PDS2的表达量。在不同组织间,Aco-PDS1和Aco-PDS2在冠芽中的表达量最高,其次是在叶、成熟果肉、未成熟果肉和根中。对Aco-PDS1进行基因编辑后在含有潮霉素的培养基上进行筛选,存活下来的愈伤组织分化成苗,绝大多数幼苗叶片呈现纯白化状态,成苗后不久便死亡。存活下来的幼苗的叶片多为浅绿色或者细线型绿色。与对照相比,这些植株生长缓慢,长势弱,叶片白化。对基因编辑株系的PAM附近序列进行PCR扩增测序,结果显示,在这些株系PAM序列附近的序列都发生了变异。变异类型有删除和插入删除。其中株系22删除最多,共删除了10个核苷酸。株系2、12、30突变结果造成了终止密码子。对突变株系叶片叶绿素a、叶绿素b、类胡萝卜素含量进行分析,结果显示,所有突变株系的叶绿素a、叶绿素b、类胡萝卜素含量都显著低于对照。株系12的色素含量最低,最高的是22。以上结果表明在这些突变体株系中,Aco-PDS1基因已经被成功进行了编辑突变,CRISPR-Cas9系统能够运用于菠萝基因编辑。For constructing geno me-editing technique for pineapple,phytoene desaturase gene was analyzed in this research.There were two phytoene desaturase genes in pineapple genome.The similarity between Aco-PDS1and PDS genes in another plants was higher than that between Aco-PDS2 and PDS genes in another plants.The expressions of Aco-PDS1 in root,leaf,crown,immature pulp,mature pulp were all higher than those of Aco-PDS2in root,leaf,crown,immature pulp,mature pulp correspondingly.Among tissues,the expressions of these two PDS genes in crown were the highest.The sequence of expressions in tissues was crown,leaf,mature pulp,immature pulp and root from high to low.Callus transformed with Aco-PDS1-edited vector was screened on medium containing hygromycin.Survived callus were differentiated into shoots.Most of the shoots were albinistic and died.Most of the survived shoots were jade green or stripy green.These shoots were lower and grow slower than control.Mutation took place in sequences near to PAM of all survied shoots.Mutations comprised deletion and insertion.Ten ribonucleotides were deleted in line 22,which has most deletions among the survived lines.Stop codons formed in line 2,12,and 30 because of mutation.Pigment contents in leaf were measured.Chlorophyll a,b and carotenoid in all of mutated lines were significantlly lower than control.Pigment content in line 12 was the lowest and line 22 was the highest.Aco-PDS1 has been mutated in these survived lines.CRISPR-Cas9 can be applied in pineapple.

关 键 词：菠萝 基因编辑 八氢番茄红素脱氢酶 

分 类 号：S668.3[农业科学—果树学]