阅读说明：本技术 梨bZIP家族新转录本PybZIPa及其重组表达载体的应用 (Pear bZIP family new transcript PybZIPa and application of recombinant expression vector thereof ) 是由 吴俊� 刘海楠 谢智华 姚改芳 张绍铃 李甲明 张明月 赵可娇 于 2019-09-29 设计创作，主要内容包括：本发明公开了梨bZIP家族新转录本PybZIPa及其重组表达载体的应用。PybZIPa转录因子核苷酸序列如SEQ ID No.1所示,其编码的氨基酸序列为序列表SEQ ID No.2所示。通过农杆菌介导的瞬时遗传转化法将PybZIPa转化烟草叶片、草莓和梨果实,经生物学功能验证,表明本发明提供的PyZIPa基因能够促进光诱导条件下的梨果皮花青苷的合成。(The invention discloses a pear bZIP family new transcript PybZIPa and application of a recombinant expression vector thereof. The nucleotide sequence of the PybZIPa transcription factor is shown in SEQ ID No.1, and the coded amino acid sequence thereof is shown in a sequence table SEQ ID No. 2. The PybZIPa is transformed into tobacco leaves, strawberries and pear fruits by an agrobacterium-mediated transient genetic transformation method, and biological function verification shows that the PyZIPa gene provided by the invention can promote synthesis of anthocyanin in pear pericarp under a photoinduction condition.)

1. A novel bZIP family transcript PybZIPa gene which is separated from a 'Mantianhong' pear and promotes the synthesis of photoinduced pericarpin in a transcription regulation mode has a nucleotide sequence shown as SEQ ID No. 1.

2. The protein encoded by the transcription factor PybZIPa gene according to claim 1, which has an amino acid sequence represented by SEQ ID No. 2.

3. A recombinant expression vector comprising the gene of claim 1.

4. The recombinant expression vector according to claim 3, wherein the gene of claim 1 is inserted between EcoR I and Xba I sites using pSAK277 as an expression vector.

5. A host bacterium comprising the gene of claim 1.

6. Use of the PybZIPa gene of claim 1 to promote anthocyanin synthesis induced by pear pericarp light.

7. Use of the recombinant expression vector of claim 3 to promote light-induced synthesis of pirifolin.

8. the use according to claim 7, characterized in that the PybZIPa gene-containing recombinant vector of claim 3 regulates the use of a anthocyanin structural gene and a promoter of an important transcription factor in promoting light-induced synthesis of hesperidin from exocarpium Citri Grandis.

Technical Field

The invention belongs to the technical field of plant molecular genetic engineering, and relates to a novel pear bZIP family transcript PybZIPa and an application of a recombinant expression vector thereof.

Background

The red-peel pear resources in Asian pear types cultivated in China are relatively few, the coloring of the red-peel pear is easily influenced by environmental factors such as illumination, temperature and the like, and the coloring influenced by the environment is unstable, so that the commercial value of the red pear is greatly influenced. The color of the red skin of pear is the result of anthocyanin accumulation, and the color type and shade are determined by the biosynthesis level and proportion of anthocyanin. Light (including photoperiod, light intensity and light quality) is an important environmental factor affecting anthocyanin biosynthesis. The effect of light on anthocyanin biosynthesis has been reported in many species: such as lichi (lichi chinensis) (Wei et al, 2011), grape (vitas L.) (Azuma et al, 2012), waxberry (Myrica rubra) (Niu et al, 2010), cowberry (Vaccinium spp.) (Uleberg et al, 2012), raspberry (Rubus ideeus) (Wang et al, 2009), and the like. In rosaceous plants, illumination also has an important significance for anthocyanin synthesis, as reported in strawberries (Kadomura-Ishikawa et al, 2013), peaches (Jia et al, 2005; Ravaglia et al, 2013), pears (Bai et al, 2017; Feng et al, 2010), apples (Sun et al, 2014; Takos et al, 2006; Takos et al, 2015; Feng et al, 2013). The research on the mechanism of photoinduced anthocyanin biosynthesis has been advanced, such as Arabidopsis thalianaCONSTITUTIVE PHOTOMOPHENIC 1(COP1), a "central regulator" of the light signal transduction pathway that inhibits photomorphogenesis in the dark, is able to interact with upstream photoreceptor proteins (e.g., CRY1, CRY2, PHYA,PHYB and UVR8), and also degradation of downstream genes by ubiquitination (Kang et al, 2009; ma et al, 2002; wu et al, 2012), ubiquitination of this gene has a significant impact on photomorphogenesis, including anthocyanin accumulation. Among the numerous downstream genes of COP1, LONG HYPOCOTYL 5(HY5, bZIP transcription factor) is directly related to photomorphogenesis, uv resistance and anthocyanin synthesis (Lau et al, 2012; Stracke et al, 2010). HY5 regulates anthocyanin biosynthesis by interacting with B-Box protein (BBXs). Furthermore, HY5 has a regulatory and regulated relationship with BBXs (An et al, 2019; Yadav et al, 2019; Heng et al, 2019; Bai et al, 2014).

Illumination affects various metabolic pathways in plants (castati et al, 2011; Stushnoff et al, 2010; Kong et al, 2016), but the regulatory network for light-induced anthocyanin biosynthesis is not clear. Thanks to the development of advanced technologies such as functional genomics, metabolomics, proteomics, and epigenetics, multi-group, combined chemical analyses have been developed in plant growth (Persson et al, 2005; Sekhon et al, 2012;et al, 2014), environmental response (Caldana et al, 2011; cho et al, 2008; tan et al, 2015) and other biological processes (Cho et al, 2016; hirai et al, 2007; gutierrez et al, 2008) were used. In particular, the association of transcriptome and metabolome directly reveals the relationship between genes and metabolites not only in Arabidopsis (Caldana et al, 2011; Hirai et al, 2007; Cohen et al, 2017; Hirai et al, 2004) but also in citrus (Citrus)et al, 2014; tan et al, 2015; lu et al, 2017), grapes (Agudelo-Romero et al, 2015; fortes et al, 2011), kiwi fruit (Liu et al, 2018), lychee (Yun et al, 2016) and fig (Wang et al, 2017). Although some progress has been made in the mechanism of anthocyanin biosynthesis, such as anthocyanin synthesis genes (e.g., ANS, DFR, UFGT, etc.) and transcription regulatory complexes (e.g., MYB-bHLH-WD40, MYB-bHLH-ERF) (Zhanget al, 2011; yao et al, 2017). However, in the anthocyanin biosynthesis process affected by illumination, the combined analysis of differentially expressed genes and differentially metabolites has been rarely reported. The invention relates to a method for mining anthocyanin synthesis key genes induced by pear light by virtue of multiomic combined analysis (transcriptome and metabolome), and defining the regulation and control action mechanism of the anthocyanin synthesis key genes, so as to provide important gene resources for pear coloring regulation and genetic improvement.

Disclosure of Invention

the invention aims to provide a transcription factor PybZIPa gene for promoting photoinduced anthocyanin synthesis.

Another purpose of the invention is to provide the application of the gene.

The purpose of the invention can be realized by the following technical scheme:

A PybZIPa transcription factor separated from 'MANTIANHONG' pear and having the function of regulating light-induced anthocyanin synthesis in pericarp is screened from a transcriptome and metabolome correlation analysis result, is a new transcript of a bZIP family, and the nucleotide sequence of the new transcript is shown as SEQID No.1 and comprises an open reading frame of 765 bp; 218 amino acids are coded, the coded amino acid sequence is shown as SEQ ID No.2, the isoelectric point is 5.58, and the molecular weight is 24.3 kDa.

a recombinant expression vector containing the PybZIPa gene of the invention.

The recombinant expression vector is preferably a pSAK277 expression vector, and the insertion site of the PybZIPa gene is between EcoR I and Xba I.

A host bacterium containing the PybZIPa gene of the present invention.

Cloning the primer pair of the PybZIPa gene cDNA sequence, wherein the sequence of an upstream primer PybZIPa-EcoR I-F is shown as SEQ ID No.3, and the sequence of a downstream primer PybZIPa-Xba I-R is shown as SEQ ID No. 4.

The PybZIPa gene disclosed by the invention is used for promoting synthesis of photoinduced pericarp anthocyanin. The PybZIPa gene can up-regulate the expression of PyUFGT by combining a G-box element connected in series on a PyUFGT promoter, and simultaneously regulate and control the expression of anthocyanin synthesis related transcription factors PyMYB114, PyMYB10 and PyBBX22 by the G-box element, and can also activate the transcription activity of the promoter per se, so that the synthesis of light-induced anthocyanin in pear pericarp is finally promoted. The PyUFGT, PyMYB114, PyMYB10, PyBBX22 and PybZIPa gene promoter sequences are shown as SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8 and SEQ ID No.9 in sequence.

The PybZIPa gene-containing recombinant expression vector is applied to promotion of synthesis of anthocyanin in pear pericarp.

Advantageous effects

Compared with the prior art, the invention has the following advantages and effects:

1. The PybZIPa gene screen is selected from the combined analysis result of 'Mantianhong' pericarp transcriptome and metabolome data, and the PybZIPa gene screen is a new transcript of the bZIP family, and the homology rate of the new transcript and the annotated anthocyanin regulation related bZIP family members is only 31-32 percent and is respectively positioned at different chromosome positions. Compared with other bZIP family members, the PybZIPa expression disclosed by the invention has stronger response to light-induced anthocyanin synthesis.

2. The PybZIPa gene disclosed by the invention is applied to promoting photoinduced synthesis of anthocyanin in pear fruits, provides a new gene resource for molecular breeding of red-peel pears, also expands a regulation and control mechanism of a luminous environment factor on synthesis of anthocyanin in pear fruits, and is beneficial to improvement of appearance quality and market value of pears by development and utilization of genetic resources.

3. Through an agrobacterium-mediated transient transformation method, the transcription factor PybZIPa promotes the accumulation of anthocyanin in tobacco leaves, pear peel and strawberry fruits, and biological function verification shows that the PybZIPa cloned by the invention has the function of promoting the synthesis of photoinduced anthocyanin in pear peel by regulating and controlling anthocyanin synthesis structural genes and important transcription regulation and control factors.

drawings

FIG. 1 is a table graph and anthocyanin composition determination for 'Mantianhong' pears bagged, de-bagged. 'Mantianhong' pears are bagged 35 days after blossom (special bags for double-layer pears with yellow outer and black inner), and are bagged 10 days before maturity. B1, B2 and B3 are the peels of the 4 th, 8 th and 10 th days after bag removal; a1, A2, A3 are the non-sacked peels at the corresponding time. A, the phenotype of the 'Mantianhong' pear after bagging and bag removal. Bagging 'Mantianhong' pear, change of anthocyanin content after bag removal and component determination.

FIG. 2 is a diagram showing the expression pattern of light response and anthocyanin metabolic pathway genes in 'Mantianhong' pear bagging and bag-removed pericarp. The heatmap showed that anthocyanin metabolic pathway genes in de-bagged peel were significantly higher than those in un-de-bagged peel.

FIG. 3 is a diagram showing the expression patterns of PybZIPa, anthocyanin structural genes and important transcription factors in the peel of the bagged and unpacked pear of Mantianhong. The anthocyanin structural gene comprises: PyCHI, PyANS, PyUFGT; important transcription factors: PyMYB114, PyMYB10 and PyBBX 22.

FIG. 4 is a graph showing the comparative analysis of subcellular localization of PybZIPa of the present invention with other family members. A. The chromosomal location and subcellular localization of PybZIPa of the present invention. B. Phylogenetic tree analysis and conserved domain analysis of transcription factors of Arabidopsis thaliana and Rosaceae fruit tree HY 5.

FIG. 5 shows PybZIPa of the present invention promoting anthocyanin accumulation and anthocyanin structural gene expression. A. A phenotype picture of tobacco leaf (I), pear peel (II) and strawberry fruit (III) coloring. B. Expression pattern of anthocyanin synthesis related gene after over-expression of PybZIPa. Note: empty vector: pSAK277 (empty) phenotype; PybZIPa OE: over-expressing the PybZIPa phenotype.

FIG. 6 shows that the PybZIPa of the present invention has a regulatory effect on anthocyanin synthesis structural genes and important transcriptional regulatory factors by using a dual luciferase and yeast single hybrid (Y1H). A. Construction of a dual-luciferase experimental vector, promoter action element analysis and dual-luciferase detection. B. And (5) yeast single hybrid result graph.

FIG. 7 is a pattern diagram of PybZIPa regulation and control of light-induced anthocyanin synthesis. After the pear is bagged, PybZIPa strongly responds to illumination, and the anthocyanin synthesis structural gene and important transcription regulation factor are regulated and controlled by combining a G-box element, and meanwhile, a promoter of the pear can be activated, so that anthocyanin synthesis induced by pear peel light is finally promoted.

Detailed Description

The present invention will be described in detail with reference to specific examples. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.