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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (8): 1146-1156.doi: 10.3724/SP.J.1006.2020.94198

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Mechanism of BnaBZR1 and BnaPIF4 regulating photosynthetic efficiency in oilseed rape (Brassica napus L.) under poor light

FENG Tao1,2,TAN Hui1,2,GUAN Mei2,GUAN Chun-Yun2,*()   

  1. 1College of Biology and Agriculture, Zunyi Normal College, Zunyi 563000, Guizhou, China
    2College of Agronomy, Hunan Agricultural University/National Oilseed Crops Improvement Center in Hunan, Changsha 410128, Hunan, China
  • Received:2019-12-16 Accepted:2020-03-24 Online:2020-08-12 Published:2020-04-03
  • Contact: Chun-Yun GUAN E-mail:guancy2011@aliyun.com
  • Supported by:
    National Basic Research Program of China (973 Program)(2015CB150206)

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Abstract:

Double-low seed rape B. napus L. varieties XY881 and XY883 screened from the same parent Xiangyou 15 have significant differences in seed oil content, photosynthetic efficiency, and poor light sensitivity. Brassinazole-resistant 1 (BnaBZR1) and phytochrome interacting factor 4 (BnaPIF4) genes were cloned from XY881 and XY883, respectively, and their sequence structure, gene expression and gene function were analyzed. There were structural mutations in the BnaBZR1 and BnaPIF4 of XY883, causing differences in gene expression and regulation. The promoter of BnaBZR1 in XY883 had a 124 bp A/T-rich insertion mutation, and XY883 had a higher expression of BnaBZR1 than XY881, and fewer expression changes under low light and 2,4-epibrassinolide(2,4-BL) treatment. Splicing differences of BnaPIF4 in the 5'-UTR region were found in XY883, and the alternative splicing resulted in three 5'-UTRs of 424 bp (U01), 239 bp (U02), and 332 bp (U03). Under the induction of low light and 2,4-BL treatments the changed of three alternative spliced BnaPIF4 transcripts in XY883 had significant differences. The three 5'-UTRs combined with the CDS of BnaPIF4 were transformed into Arabidopsis thaliana. There was no significant difference in BnaPIF4 gene expression, but a significant difference in protein expression, indicating that the 5'-UTR mutation of BnaPIF4 affects protein synthesis. Transgenic BnaPIF4 Arabidopsis thaliana showed a phenotype with increased plant height, narrow leaves, and decreased photosynthesis. Co-transformation whit BnaBZR1 could attenuate the decrease in photosynthesis caused by BnaPIF4. The effects of BnaPIF4 and BnaBZR1 genes on Brassica napus L. were similar to those on Arabidopsis thaliana, while the phenotype was not as obvious as that of Arabidopsis thaliana, suggesting that BnaPIF4 is a negative regulator of photosynthesis in rapeseed, and BnaBZR1 can antagonize the negative regulation of photosynthesis by BnaPIF4, while is consistent with the regulation pattern and photosynthetic phenotype of the two genes in XY881 and XY883.

Key words: Brassica napus L., BnaPIF4, BnaBZR1, alternative splicing, insertion mutation

Table 1

Primer for gene cloning"

引物
Primer		 克隆产物
Product of cloning		 序列
Sequence (5'-3')
PIF4_mF mRNA of BnaPIF4 ATAGATCTCATCCCTAAAGA
PIF4_mR TATGTTCAAAAGATAGCCTTAG
BZR1_mF mRAN of BnaBZR1 GGAGAAGGAAAGAGAGATTC
BZR1_mR TTGAGAGAAACAAAATGGGC
PIF4_cF CDS of BnaPIF4 ATGGAACACCAAGGTTGGAG
PIF4_cR CTAACGGGGACCGTCGG
BZR1_cF CDS of BnaBZR1 ATGACGTCAGATGGAGCTACG
BZR1_cR TCAACCACGAGCTTTGCC
PIF4_pF Promoter of BnaPIF4 ATTGAAACCGATTGTAAGGA
PIF4_pR AGAAACAAAGGAGCATAAAG
BZR1_pF Promoter of BnaBZR1 GGTTATTTTCAAATAATGGATG
BZR1_pR CTTGAGCTCTTAGCCCTGTG
PIF4_uF 5'-UTR of BnaPIF4 ATAGATCTCATCCCTAAAGA
PIF4_uR GTCAGATCTCAGATTTGGAAAGC
PIF4_dF Full-length gene of BnaPIF4 ATAGATCTCATCCCTAAAGAAG
PIF4_dR TTTTGACAAACTAAACCAGG

Table 2

Primer for RT-qPCR"

引物
Primer		 基因
Gene		 序列
Sequence (5'-3')
ActF BnaActin GGTTGGGATGGACCAGAAGG
ActR TCAGGAGCAATACGGAGC
PIF4F BnaPIF4_cds CTGTGCTGTTGTGCTTAC
PIF4R AGTCTCTACATAAACCCATAGG
U1_F BnaPIF4_UTR_U01 CTGTGCTGTTGTGCTTAC
U1_R AGTCTCTACATAAACCCATAGG
U2_F BnaPIF4_UTR_U02 CTGTGCTGTTGTGCTTAC
U2_R TTCCTCTCACTTGCTCTC
U3_F BnaPIF4_UTR_U03 GAGAGCAAGTGAGAGGAA
U3_R CAGAAGCTGAAGTAGTAGAAG
BZR1F BnaBZR1 CTCTCATCTCCAACTTCCAA
BZR1R GACTCATCACACTCAGGTATA

Fig. 1

Investigation of important agronomic traits of XY881 and XY883 under poor light and 2,4-BL treatment A: oil content of seed; B: oleic acid content of seed; C: total number of silique per plant; D: number of seeds per silique; E: 1000-seed weight of mature seeds; F: RuBPCase content in leaves. *P < 0.05, **P < 0.01."

Fig. 2

Different gene structures of BnaPIF4 and BnaBZR1 in XY881 and XY883 A: cloning of mRNA of BnaPIF4 and BnaBZR1; B: cloning of CDS of BnaPIF4 and BnaBZR1; C: cloning of promoter of BnaPIF4 and BnaBZR1; D: cloning of 5'-UTR of BnaPIF4; E: cloning of full-length BnaPIF4 gene; F: the mutation structure of BnaPIF4 and BnaBZR1 in XY881 and XY883. 1: BnaPIF4 of XY881; 2: BnaPIF4 of XY883; 3: BnaBZR1 of XY881; 4: BnaBZR1 of XY883; 01-03: three 5'-UTRs of BnaPIF4 from XY883; M1: 2K plus DNA marker; M2: 2K DNA marker."

Fig. 3

Insertion mutation in the BnaBZR1 promoter changed the BnaBZR1 gene expression, did not change the interaction of BnaBZR1 with BnaPIF4 A: sequence analysis of insertion mutation regions in the promoter of BnaBZR1; B: analysis of the interaction between BnaBZR1 and BnaPIF4 by yeast hybridization; C: the temporal and spatial gene expression of BnaBZR1 in XY881 and XY883; D: the expression of BnaBZR1 in XY881 and XY883 under poor light stress and 2,4-BL treatment. DAG means days after seed germination."

Fig. 4

Alternative splicing affects BnaPIF4 gene expression and protein translation A: temporal and spatial gene expression of BnaPIF4 in XY881 and XY883; B: the expression of BnaPIF4 in XY881 and XY883 under poor light stress and 2,4-BL treatment; C: construction of pRI101-AN vector with different 5'-UTRs; D: the mRNA expression of BnaPIF4 and the protein expression of BnaPIF4; E: the phenotype of transgenic BnaPIF4 Arabidopsis thaliana; F: the RuBPCase protein content of transgenic BnaPIF4 Arabidopsis thaliana. DAG means days after seed germination."

Fig. 5

Phenotype of transgenic Arabidopsis thaliana and B. napus L. A: the phenotype of transgenic BnaPIF4 and BnaBZR1 Arabidopsis thaliana; B: the RuBPCase protein content of transgenic BnaPIF4 and BnaBZR1 Arabidopsis thaliana; C: the oil content of transgenic Arabidopsis thaliana seed; D: the plant of transgenic B. napus L.; E: the gene expression of BnaPIF4 and BnaBZR1 in transgenic B. napus L.; F: the RuBPCase content of leaves of transgenic B. napus L."

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