Cloning and Expression Analysis of CaMADS6 in Cytoplasmic Male Sterile Line and Maintainer Line of Pepper

doi:10.7668/hbnxb.20193440

Abstract

Abstract:

To explore the relationship level between CaMADS6 expression and floral organ development of pepper,CaMADS6 was cloned for bioinformatics prediction from the sterile line 9704A and the maintainer line 9704B,and the spatiotemporal expression characteristics of this gene were analyzed.The results showed that the CaMADS6 cloned from 9704A and 9704B had the same coding sequence,with a full length of 744 bp and encoding 247 amino acid residues.CaMADS6 protein had the relative molecular weight of 28.67 ku and theoretical isoelectric point of 8.98.It was a hydrophilic protein with no transmembrane structure.The secondary structure of CaMADS6 consisted of 57.49% α-helices,8.91% extended chains and 28.74% irregular coils.The CaMADS6 protein shared 100% homology with CaFUL2,which possessed a typical MADS-box characteristics.CaMADS6 was expressed in different organs of 9704A and 9704B at different developmental stages and the expression was the highest in flower buds,followed by leaves and stems,and almost no expression in roots.The expression of CaMADS6 in stems of 9704A was significantly lower than that of 9704B at seedling,flower bud and adult stages,and the expression of CaMADS6 in leaves was significantly lower than that of 9704B at the adult stage.The expression level of CaMADS6 in flower buds of 9704A was extremely significantly higher than that of 9704B at flower bud and adult stage,and its expression level was 2.2 and 3.5 times higher than in 9704B,respectively;CaMADS6 gene was expressed in different parts of flower organs of 9704A and 9704B,and the expression of CaMADS6 gene from high to low was calyx,corolla,ovary and anther.The expression level of CaMADS6 in the calyx and corolla of 9704A was extremely significantly or significantly lower than that of 9704B,the expression level of CaMADS6 were 66%,83% of 9704B,respectively.The expression level of CaMADS6 in 9704A anther was 34 times higher than in 9704B,and the difference between the two groups was extremely significant,predicting the abnormal expression of CaMADS6 in anthers was closely related to pepper male sterility.

Key words: Pepper, Male sterility, CaMADS6, Gene cloning, Expression analysis

LIAO Mingyu, XIAO Jialin, LI Liyuan, SONG Yu, HUANG Hurong, YANG Bozhi. Cloning and Expression Analysis of CaMADS6 in Cytoplasmic Male Sterile Line and Maintainer Line of Pepper[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(1): 46-52. doi: 10.7668/hbnxb.20193440.

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Figures/Tables 7

Fig.1 The amplification bank of CaMADS6 gene M.5 kb DNA ladder。

Tab.1 The amino acid composition and proportion of CaMADS6

氨基酸组成 Amino acid composition	氨基酸数目 Number of amino acids	所占比例/% Ration	氨基酸组成 Amino acid composition	氨基酸数目 Number of amino acids	所占比例/% Ration	氨基酸组成 Amino acid composition	氨基酸数目 Number of amino acids	所占比例/% Ration
Ala	13	5.3	Arg	19	7.7	Asn	17	6.9
Asp	9	3.6	Cys	2	0.8	Gln	22	8.9
Glu	23	9.3	Gly	13	5.3	His	11	4.5
Ile	7	2.8	Leu	28	11.3	Lys	17	6.9
Met	7	2.8	Phe	5	2.0	Pro	7	2.8
Ser	21	8.5	Thr	7	2.8	Trp	3	1.2
Tyr	6	2.4	Val	10	4.0

Fig.2 The coding region and its corresponding amino acid sequences of CaMADS6

Fig.3 Alignment of amino acid sequences between CaMADS6 of pepper and FUL2 of other species The red and black line segments indicate MADS_MEF2_like structural domain consisting of 78 amino acids(residues 2—79)and the K-box structural domainconsisting of 84 amino acids(residues 90—173),respectively;the blue shading indicates different amino acid residues and black shading indicates identical amino acid residues.CaMADS6.Capsicum annuum (AF130118.2);CaFUL2.Capsicum annuum (NM_001324623.2);SlFUL2.Solanum lycopersicum (NM_001307938.1);BsFUL2.Brugmansia suaveolens (MT069983.1);PpFUL2.Physalis pubescens (KC414862.1);NtFUL2.Nicotiana tabacum (NM_001325205.1);WsFUL2.Withania somnifera (MH931169.1);SjFUL2.Streptosolen jamesonii (MH931166.1);CnFUL2.Cestrum nocturnum (MT069971.1);PeFUL2.Petunia exserta (MH931145.1).

Fig.4 Phylogenetic tree analysis of amino acid sequences of CaMADS6 in pepper and FUL in other species TwFUL-L.Tripterygium wilfordii (NC_052235.1);PvFUL-L.Pistacia vera (NC_048444.1);NtFUL-L.Nicotiana tomentosiformis(NW_008885358.1);CiFUL-L.Carya illinoinensis (NC_056754.1);QlFUL-L.Quercus lobata(NC_044905.1);RcFUL-L.Rosa chinensis(NC_037089.1);VrFUL-L.Vitis riparia(Riverbank grape)(NC_048444.1);VvFUL-L.Vitis riparia (Wine grapes)(NC_012020.3);GhFUL-L.Gossypium hirsutum(Cotton)(NC_053443.1);MeFUL-L.Manihot esculenta(Cassava)(NC_035161.2);EgFUL-L.Eucalyptus grandis(NC_052612.1);SsFUL-L.Salvia splendens(NC_056042.1);NtFUL1.Nicotiana tomentosiformis(NW_015899026.1);SlFUL1.Nicotiana tabacum(Common tobacco)(NC_015443.3);StFUL1.Solanum tuberosum(Potato)(NW_006239107.1);InFUL1.Ipomoea nil (Japanese morning glory)(NW_017618323.1);SoFUL.Spinaciaoleracea(Spinach)(NW_018932157.1);BnFUL.Brassica napus(Rape)(NW_019168750.1);AtFUL.Arabidopsis thaliana(Thale cress)(NC_003076.8);BoFUL-a.Brassica oleracea(Wild cabbage)(NC_027749.1);BoFUL-b.Brassica oleracea(Wild cabbage)(NC_027754.1);BoFUL-c/BoFUL-d.Brassica oleracea(Wild cabbage)(NC_027756.1).

Fig.5 The relative expression of CaMADS6 in the different organs at different stages of 9704A and 9704B The expression of CaMADS6 gene in stems of 9704A seedling was used as the control for analysis of variance.The different lowercase letters indicated significant difference P<0.05 and the different capital letters indicated extremely significant difference P<0.01.The same as Fig.6.

Fig.6 The relative expression of CaMADS6 gene in different parts of floral organs of 9704A and 9704B The expression of CaMADS6 gene in anthers of 9704A was used as the control for analysis of variance.

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