Cloning and Expression Analysis of Maize PLATZ Gene GRMZM2G006585 Promoter

doi:10.7668/hbnxb.20193576

Abstract

Abstract:

Grain specific promoter with high transcription activity can regulate the specific and high-level expression of target genes in plant grains.In order to explore specific promoters of maize grain,the dominant expression gene GRMZM2G006585 was screened from the published data of maize expression profile microarray.The DNA sequence about 2 000 bp upstream of its coding region was cloned and named PZm2G006585.By using the online websites New PLACE and PlantCARE to analyze its promoter elements,we found that it contained multiple grain specific related elements such as E-box and P-box.It was preliminarily considered that the upstream sequence of the cloned coding region was a grain specific promoter from maize.In order to verify its function,the expression vector of GUS gene was constructed and transformed into plants.GUS histochemical staining results of transgenic rice showed that the expression pattern of exogenous genes driven by the promoter was grain specific and embryo dominant.The results of GUS activity detection in T₃ seeds of transgenic Arabidopsis thaliana single copy line showed that the GUS activity driven by PZm2G006585 was 909.52 nmol/(min·mg).The discovery and functional verification of the grain-specific promoter PZm2G006585 can provide candidate promoter resource for specific expression of target genes in maize,rice and other monocotyledons.

Key words: Maize, Grain specific promoter, Plant bioreactor, PZm2G006585, Genetic transformation

WANG Yali, WEI Qichao, LI Chengwei. Cloning and Expression Analysis of Maize PLATZ Gene GRMZM2G006585 Promoter[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 21-30. doi: 10.7668/hbnxb.20193576.

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Figures/Tables 11

Tab.1 Primers for expression profile analysis

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
ZmTubulin_RT(F)	CCATCTATGACATCTGCCGCCGCTC
ZmTubulin_RT(R)	CAGGGAAGCCGTCAGGGATGAGATG
ZmGAPDH_RT(F)	ACCACCAACTGCCTTGCTCCAC
ZmGAPDH_RT(R)	TTGTGTACTTCTTCCCTTAGCGATGT
GRMZM2G006585_RT(F)	AATGGTATGGATCATGTGGCTAGG
GRMZM2G006585_RT(R)	TTTGGTGGTGGTTCTTCGTAGTT

Tab.2 Real time fluorescence quantitative PCR primers

引物名称 Primer name	引物序列(5'-3') Primer sequence(5'-3')
ZmLeg1_RT(F)	GTGACCAGCGAGGCGAGATC
ZmLeg1_RT(R)	GACTTGTGACGATGGGAACG
ZmZein27_RT(F)	GATCTTCGGCTTGGTCCTCC
ZmZein27_RT(R)	CGTCAGTTGCTGCGCTATCT

Fig.1 Transcriptional abundance of GRMZM2G006585 in different tissues(organs)detected by RT-PCR R,ST,L,A,PO,H,SI,C and G respectively represent root,stem,leaf, anther,pollen,husk,silk,cob and grain at 18 days after pollination.

Fig.2 Relative transcript abundance of GRMZM2G006585 at different stages of grain development detected by qRT-PCR DAP3,6,9,12,18,24,32 represent 3,6,9,12,18, 24,32 days after pollination,respectively.

Fig.3 Cloning of upstream sequence of GRMZM2G006585 coding region M.DNA standard molecular weight DL5000+; 1.PZm2G006585.

Fig.4 Promoter cis-acting element analysis of upstream sequence of GRMZM2G006585 coding region Cis-acting elements are indicated by underscores,and the transcription site is indicated by +1.

Fig.5 GUS staining of multiple organs in T₀ generation of transgenic rice A.Root; B.Stem; C.Leaf;D.Flower. 1.Pubi; 2.Promoter deletion;3.PZm2G006585; 4.POsGluB-4; 5.PZmZein27; 6.PZmLeg1.

Fig.6 GUS staining in different developmental stages of T₁ generation grains of transgenic rice 1.Pubi;2.缺失启动子;3.POsGluB-4;4.PZmZein27;5.PZmLeg1;6.PZm2G006585。 A.Testa(3 days after flowering, DAF 3); B.Caryopsis(DAF 7);C.Testa(DAF 7); D.Caryopsis (DAF 12); E.Testa(DAF 12); F.Caryopsis(DAF 40); G.Testa(DAF 40).1.Pubi; 2.Promoter deletion; 3. POsGluB-4; 4.PZmZein27; 5.PZmLeg1; 6.PZm2G006585.

Fig.7 Expression pattern of PZm2G006585 in rice grains A1.Promoter deletion(DAF 40); A2. PZm2G006585(12 days after flowering, DAF 12); A3.PZm2G006585(DAF 20); B1.PZm2G006585(DAF 30); B2.PZm2G006585(DAF 35); B3.PZm2G006585(DAF 40).

Fig.8 Screening of single copy homozygous Arabidopsis A.Screening of transgenic plants;B.PCR identification of GUS in some T₁ generation positive strains(M.DNA Marker; 1-47.Serial number of transgenic Arabidopsis;48.Negative control);C.T₃ generation single copy homozygotic seedlings.

Fig.9 GUS activity analysis in Arabidopsis Different letters represent significant difference at the 0.05 level.

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