芥蓝miR156a家族进化特性及表达分析

Evolutionary Characteristics and Gene Expression Analysis of miR156a Family in Chinese Kale

miRNA广泛参与植物的发育过程。芥蓝形态多样,叶片发育因品种而异。为了解miR156a家族的进化特性及其在芥蓝叶片发育中的表达模式。该研究对芥蓝miR156a成员及其前体pre-miR156a成员进行生物信息学分析,比较不同品种芥蓝叶形的差异,并采用qRT-PCR方法分析芥蓝不同组织部位中pre-miR156a的表达水平、以及叶形相似的芥蓝品种‘翠宝’和‘改良香菇’中不同类型叶片的pre-miR156a成员及其靶基因的表达水平。结果表明:(1)多序列比对和进化树分析发现芥蓝miR156a家族成员和pre-miR156a-3p_1在进化过程中高度保守;二级结构预测发现pre-miR156a每个成员均能形成茎环结构并包含2~3个miR156a成员序列;靶基因预测显示miR156a-5p和miR156a主要靶向SPL,而miR156a-3p_1则靶向CTPS等不同的基因。(2)‘翠宝’和‘改良香菇’芥蓝的叶形最为相似,qRT-PCR分析显示,pre-miR156a在‘翠宝’营养生长期的叶片中高度表达。(3)pre-miR156a成员在‘翠宝’和‘改良香菇’不同类型叶片中差异表达,pre-miR156a主要在成熟叶和菇叶中表达,而pre-miR156a-3p_1和pre-miR156a-5p在第一片真叶中表达量更高。(4)靶基因分析的结果在不同品种中呈现不同趋势,‘翠宝’成熟叶中SPL10/15高表达,SPL2表达量降低;‘改良香菇’中SPL10在成熟叶和菇叶中表达降低,SPL15在菇叶中高表达,SPL2在不同类型叶片中表达无差异。研究认为,miR156a成员及其靶基因SPL2/10/15可能参与调控芥蓝叶片发育,不同品种中作用的靶基因可能存在差异,从而导致了各品种芥蓝叶片发育的差异。

miRNA is widely involved in the development of plants.The leaf type varied among different Chinese kale varieties.To understand evolutionary characteristics of the miR156a family and its expression pattern during leaf development of Chinese kale,we performed bioinformatics analysis on different miR156a members and the corresponding precursor pre-miR156a genes in Chinese kale.Morphology of leaves of four Chinese kale varieties were recorded,and expression of miR156a precursors were analyzed by qRT-PCR in different tissues of‘Cuibao’.In addition,the expression levels of pre-miR156a and SPL2/10/15 in the first true leaves,mature leaves and‘Gu’leaves were also measured in‘Cuibao’and‘Gailiangxianggu’.The results contained four aspects as below.(1)Multiple sequence alignment and evolutionary tree analysis showed that miR156a family and pre-miR156a-3p_1 was highly conserved in Chinese kale during evolution.Secondary structure prediction revealed that each member of pre-miR156a could form a stem-loop structure and contained two or three miR156a’s sequences.Target gene prediction showed that miR156a-5p and miR156a mainly targeted SPL family while miR156a-3p_1 targeted different genes such as CTPS.(2)The leaf shape of‘Cuibao’and‘Gailiangxianggu’are the most similar and the difference is that‘Gailiangxianggu’has its unique‘Gu’leaves.The qRT-PCR analysis exhibited that the expression level of pre-miR156a was high in the leaves of‘Cuibao’at vegetative growth stage.(3)The expression level of pre-miR156a members varied in different types of leaves in‘Cuibao’and‘Gailiangxianggu’.The expression of pre-miR156a was abundant in mature leaves and‘Gu’leaves,while the expression levels of pre-miR156a-3p_1 and pre-miR156a-5p were higher in the first true leaves.(4)Target gene analysis showed their different expression trends existed in different varieties.In‘Cuibao’,the expression level of SPL10/15 was high in the mature leaves while the expression level of SPL2 was reduced.In‘Gailiangxianggu’,the expression level of SPL10 was reduced in the mature leaves and‘Gu’leaves while SPL15 was high in the‘Gu’leaves and no difference was detected in the transcript amounts of SPL2.As a result,members of miR156a and their target genes SPL2/10/15 may be involved in regulating leaf development of Chinese kale.The target genes of miR156a in different varieties may be different,which might related to the various leaf phenotypes in Chinese kale.