Scientia Agricultura Sinica ›› 2018, Vol. 51 ›› Issue (24): 4690-4699.doi: 10.3864/j.issn.0578-1752.2018.24.009

• HORTICULTURE • Previous Articles     Next Articles

Apple MdMYB32 Inhibits the Anthocyanin Biosynthesis by Its Own EAR Inhibitory Sequence

XU HaiFeng(),YANG GuanXian,WANG YiCheng,JIANG ShengHui,WANG Nan,CHEN XueSen()   

  1. College of Horticulture Science and Engineering, Shandong Agricultural University/State Key Laboratory of Crop Biology, Tai’an 271018, Shandong
  • Received:2018-06-22 Accepted:2018-07-16 Online:2018-12-16 Published:2018-12-16

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Abstract:

【Objective】 In order to improve the metabolic mechanism of anthocyanin synthesis, several aspects of apple MdMYB32 in MYB transcription factors were studied, including the bio-informatics, the expression level and the function in the anthocyanin synthesis.【Method】 We cloned the MdMYB32 in Malus sieversii f. neidzwetzkyana F1 population and analyzed its phylogenetic tree and protein sequence. The expression level of MdMYB32 in different apple fruits with different stress treatments was studied. We verified its function in anthocyanin biosynthesis by transgene and analyzed its interaction by yeast one-hybrid.【Result】 qRT-PCR analysis showed that the expression level of MdMYB32 in 'Hongcui No. 9' with high anthocyanin content was lower than that in 'Hongcui No. 6' with low anthocyanin content. The expression level of MdMYB32 was negatively correlated with the content of anthocyanin. Both salt stress and cold stress could inhibit the expression of MdMYB32. The phylogenetic tree indicated that MdMYB32, AtMYB32, MdMYB16 and AtMYB4 were located in the same evolutionary branch, and MdMYB32 protein contained an EAR inhibitory sequence at the C-terminus. Overexpressing MdMYB32 in red-fleshed callus could inhibit the expression of ANS and reduce the anthocyanin content. However, when we overexpressed LESMdMYB32 (knocked out the EAR sequence of MdMYB32) in red-fleshed callus, we found that it could not affect the expression of ANS and the anthocyanin content. Yeast one-hybrid and Chip-PCR analyses showed that MdMYB32 and LESMdMYB32 could bind the promoter of ANS.【Conclusion】 MdMYB32 could bind the promoter of ANS and inhibit the anthocyanin biosynthesis by its own EAR inhibitory sequence.

Key words: apple, MYB transcription factors, MdMYB32, EAR inhibitory sequence, yeast one-hybrid

Fig. 1

Sectional drawing of apples in the mature period"

Fig. 2

Relative content of anthocyanin in 3 apple strains"

Fig. 3

Relative expression of structural genes associated with anthocyanin synthesis and related transcription factors in 3 apple strains"

Fig. 4

Phylogenetic tree of MYB32 and related MYB protein"

Fig. 5

Sequence analysis of MYB32 and related protein"

Fig. 6

Relative expression of MdMYB32 treated with cold stress or salt stress"

Fig. 7

Red-fleshed callus and the red-fleshed callus that overexpressing MYB32 or LESMYB32"

Fig. 8

Relative content of anthocyanin in 3 kinds of callus"

Fig. 9

Relative expression level of related genes involved of anthocyanin biosynthesis in 3 callus"

Fig. 10

Yeast one-hybrid analysis in MdMYB32, LESMdMYB32 and MdANS promoter"

Fig. 11

Chip-PCR analysis in MdMYB32, LESMdMYB32 and MdANS promoter"

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