CN114836440A

CN114836440A - Rice leaf color regulation gene AF1 and application thereof

Info

Publication number: CN114836440A
Application number: CN202210630435.9A
Authority: CN
Inventors: 张婷; 凌英华; 肖文文; 游静; 桑贤春; 何光华
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2022-06-06
Filing date: 2022-06-06
Publication date: 2022-08-02
Anticipated expiration: 2042-06-06
Also published as: CN114836440B

Abstract

The invention belongs to the technical field of rice breeding, and particularly relates to a rice leaf color regulating gene AF1 and application thereof. The invention provides a rice leaf color regulation gene AF1, the nucleotide sequence of which is shown as SEQ ID No.1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2. The gene coding protein is located in chloroplast, and the regulation and control of the color of rice leaves are realized by the PSI assembly regulated and controlled by interaction with PYG7, so that the gene coding protein can be used as a marker for rice molecular breeding and has important significance for the rice molecular breeding.

Description

Rice leaf color regulation gene AF1 and application thereof

Technical Field

The invention belongs to the technical field of rice breeding, and particularly relates to a rice leaf color regulating gene AF1 and application thereof.

Background

Leaves are an important place for photosynthesis of green plants, and mutation of leaf color usually changes chlorophyll content, affects photosynthesis rate, changes plant yield and even causes plant death. At present, researches show that the development of rice leaf color is regulated and controlled by different pathways. Firstly, the chlorophyll synthesis and degradation pathway is blocked, which can affect the leaf color development of rice. OsCAO1 and OsCAO2 encode chlorophyll a oxygenase, and the mutation of OsCAO1 gene can block the synthesis of chlorophyll b, so that the color of the leaf is lightened; YGL1 encodes chlorophyll synthase, and the mutation of this gene causes chloroplast hypoplasia and delayed thylakoid membrane formation, resulting in abnormal yellow-green color of leaves. Mg chelatase is a key enzyme in the chlorophyll synthesis process, OsCHLH is the first cloned gene in rice, and encodes the H subunit of Mg chelatase, and the gene causes plant albinism after mutation; LYL1 encodes geranyl reductase, which participates in the last step of chlorophyll synthesis in rice, and the mutant has yellow leaf phenotype. The NYC1 gene is a gene necessary for chlorophyll degradation and encodes a chlorophyll b reductase, and the mutation of the gene makes the NYC1 mutant keep green during the senescence period. Secondly, the leaf color development of rice is also influenced by the differentiation and development obstruction of chloroplasts. The V4 gene encodes a PPR protein located in chloroplast, and the V4 gene is mutated by low-temperature stress, so that the chloroplast development is damaged, and the V4 mutant plant is yellow. The YSA gene encodes a PPR protein located in chloroplast, contains 16 PPR motifs connected in series, has an important effect on controlling the development of chloroplast, and mutation of the PPR motif can cause albinism of rice seedlings. Kusumi et al identified early albino mutant v1 of rice, and the gene codes chloroplast localization protein NUS1, NUS1 participates in the metabolic regulation process of chloroplast RNA, and plays an important role in the transcriptional regulation process of ribosomal RNA. Thirdly, the obstruction of chloroplast protein transport is also associated with the development of rice leaf color. The OsABCI7 gene encodes an ABC transporter positioned in chloroplast, and due to mutation of the gene, rice cnl1 mutant plants mainly show the characteristic of leaf chlorosis. In addition, impaired cytoplasmic nuclear signaling pathways often affect leaf color development. Kong et al identified a yellow-green leaf mutant YGL8, YGL8 gene encoding a catalytic subunit of magnesium protoporphyrin IX monoester cyclase, magnesium protoporphyrin IX being an intermediate product of tetrapyrrole synthesis, one of the pathways for feedback of nuclear gene expression. Rey (k2) encodes a phosphofructokinase B-type chloroplast protein involved in nuclear cytoplasmic signaling, and the mutation thereof causes yellowing of rice leaves.

Although many rice leaf color regulatory genes have been cloned, chlorophyll metabolism, photosynthesis, photomorphogenesis, and cell senescence and death are extremely complex processes, and are regulated by nuclear genes and the plastid genome of a host. Therefore, the excavation of new rice leaf color mutants and the cloning of the regulatory genes thereof have important significance for disclosing molecular mechanisms in the aspects of chlorophyll metabolism, photosynthesis, photomorphogenesis, cell senescence, cell death and the like; meanwhile, the gene can be used as an excellent germplasm resource and a screening marker, and plays an important role in breeding.

Disclosure of Invention

In view of the above, the present invention aims to provide a rice leaf color regulatory gene AF1 and an application thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a rice leaf color regulating gene AF1, the nucleotide sequence of which is shown as SEQ ID No.1, and the amino acid sequence of the coded protein of which is shown as SEQ ID No. 2.

The invention also relates to a recombinant expression vector constructed by the rice leaf color regulatory gene AF 1.

The invention also relates to a reverse sequence and a complementary sequence of the rice leaf color regulatory gene AF 1.

The invention also relates to a recombinant expression vector constructed by the rice leaf color regulatory gene AF1 and AF1, an AF1 reverse sequence and application of an AF1 complementary sequence in rice molecular breeding.

The invention process of the application is as follows: the inventor selects a leaf color development mutant af1 from an EMS-induced indica rice west nong 1B mutant library, shows a phenotype that the leaf color of the whole growth period is lightened, and the chlorophyll content is extremely lower than that of a wild type. The results of experimental treatments at different temperatures indicate that af1 is a low temperature sensitive mutant. The thylakoid structure in chloroplast of af1 leaf was observed by transmission electron microscopy to be different from that of wild type. Map-based cloning and complementation verification results show that LOC _ Os05g45030 is a target gene of AF1 and is positioned in chloroplast, a nucleotide sequence of the LOC _ Os05g45030 is shown as SEQ ID No.1, an amino acid sequence of an encoded protein is shown as SEQ ID No.2, and a C base to T base substitution is carried out on a 3' UTR region of the LOC _ Os 45030, so that the stability of mRNA of the LOC _ Os is reduced. By amino acid sequence analysis, it was shown that AF1 encodes a PSI (photosystem I) assembly protein. AF1 and PYG7 are found to interact through membrane system yeast double-hetero and double-molecular fluorescence complementation experiments and Pull-down experiments, so that the interaction plays an important role in the assembly of PSI.

The invention has the beneficial effects that: the invention provides a rice leaf color regulation gene AF1, the nucleotide sequence of which is shown as SEQ ID No.1, and the coding protein is shown as SEQ ID No. 2. The gene is located in chloroplast, the regulation and control of the color of rice leaves are realized by the PSI assembly regulated and controlled by interaction with PYG7, and the gene can be used as a marker for rice molecular breeding and has important significance for the rice molecular breeding.

Drawings

FIG. 1 shows the phenotype and chlorophyll content of wild type and af1 over different periods of time; wherein, A-C is the phenotype of wild type and af1 in seedling stage, tillering stage and mature stage; D-F is chlorophyll content determination of wild type and af1 in three stages;

FIG. 2 shows the phenotype and chlorophyll determination of wild type versus af1 at different temperatures; wherein, A is the comparison of wild type and af1 phenotype under different temperature treatment; B-E is chlorophyll content measurement at different temperatures;

FIG. 3 shows mesophyll cell structures of the seedling-stage mutant af1 and Wild Type (WT);

FIG. 4 is a map-based clone of the AF1 gene; a is the fine positioning of AF1 gene; b is complementary transgenic plant phenotype and leaf transmission electron microscope observation; c is a partial sequence of the complementary transgenic plant containing the mutation site; d is chloroplast content determination;

FIG. 5 shows that mutations in the 3' UTR lead to a decrease in the stability of AF1 mRNA; wherein A is the expression level of AF1 in wild type and mutant leaves; b is PSA3 protein content in wild type and mutant; C. d is luciferase experiment verification and detection of the transcription activity of wild type and mutant 3' UTR; E. f is normal AF1 cDNA overexpression vector transferred into wild type and mutant transgenic plants and chlorophyll content corresponding to the transgenic plants;

FIG. 6 is an analysis of expression pattern of AF1 gene; wherein, A is the expression of AF1 in different tissues; B-C is the expression of AF1 in leaves; d is the expression of AF1 at a low temperature of 4 ℃ for different time periods; e is in situ hybridization analysis of AF1 in leaf; SB denotes the base of the shoot apex; l4 denotes a fourth leaf; l2 denotes a second leaf; L3L denotes the lower base of the third leaf half; L3U denotes the upper half of the third leaf;

FIG. 7 is an analysis of AF 1-encoded protein; wherein, A is AF1 evolutionary tree analysis; b is GFP signals of AF1 fusion proteins with different lengths constructed in rice protoplasts; AF1 includes 271 amino acids in full length;

FIG. 8 is an AF1 and PYG7 interaction; wherein A is membrane system yeast double-hybrid verification AF1 and PYG7 interaction; pBT3-STE-AF1 and pOst1-NubI as positive controls, pBT3-STE-AF1 and pPR3-N as negative controls; b is bimolecular fluorescence complementation experiment (BiFC) verification AF1 and PYG7 interaction; c is the interaction of Pull-down experimental verification AF1 and PYG 7; d is PYG7 tissue-specific expression analysis;

Detailed Description

The present invention will now be described more fully hereinafter with reference to the accompanying specific embodiments, in which some, but not all embodiments of the invention are shown. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments disclosed herein are intended to be within the scope of the present invention.

First, mutant discovery and phenotypic characterization

(1) Discovery of mutants

The applicant screened a leaf color development mutant af1 from EMS-induced indica rice west nong 1B mutant library. The wild type plant shows green leaves in the normal growth and development process, and compared with the wild type plant, the mutant shows that the leaves in the whole growth period are lightened in color and are yellow green.

(2) Study analysis of chlorophyll content

The photosynthetic pigment content can directly or indirectly influence the color of plant leaves, and since the af1 mutant is yellow green in the whole growth period, the chlorophyll content of the leaves of the wild type and the af1 mutant is respectively measured in the seedling stage, the tillering stage and the heading stage. The results show that the chlorophyll a, chlorophyll b, total chlorophyll content and carotenoid content of the mutant af1 leaf blade were significantly reduced compared to the wild type at each stage of the assay (P <0.01), indicating that the af1 mutant yellow-green phenotype leaf is caused by a reduction in its leaf photosynthetic pigment content (fig. 1).

(3) Phenotypic expression and relationship study of chlorophyll content to temperature

Further, the relationship between the phenotypic expression and the chlorophyll content and the temperature was investigated at 20 ℃, 25 ℃ and 30 ℃. The results show that the color of the mutant leaves is not much different from that of the wild type leaves at the temperature of 30 ℃, but the phenotype of the mutant is changed into more obvious light green leaves as the temperature is reduced. In addition, chlorophyll a, chlorophyll b, carotenoids and total chlorophyll content were determined. The results show that the chlorophyll content of the mutant is lower than that of the wild type along with the temperature reduction. Thus, it was concluded that af1 was induced by low temperature and was a low temperature-sensitive leaf color mutant (FIG. 2).

(4) Observation and study of transmission electron microscope

To further analyze whether the leaf yellowing phenomenon caused by AF1 deletion is caused by the blocked development of leaf chloroplast, ultrathin sections of leaves with the same leaf age and leaf position between the seedling stage mutant and the corresponding wild type were respectively observed by a transmission electron microscope (FIG. 3). As a result, no significant difference in the number of leaf chloroplasts was found between the mutant and the wild type. However, compared with the thylakoid basal lamina structure with clear and closely stacked structure in the chloroplast of the wild type leaves, the thylakoid basal lamina structure in the chloroplast of the mutant leaves is loose, the number of stacked basal lamina layers is obviously reduced, and the basal lamina structure becomes vague. Furthermore, the osmyl bodies of the chloroplasts of the mutant leaves were significantly increased compared to the wild type. It is shown that AF1 plays an important role in regulating the biosynthesis and basal lamina layer construction of chloroplast thylakoid membranes. The AF1 deletion causes the chloroplast thylakoid basal lamina membrane structure defect, and further causes the leaf yellowing phenomenon.

Second, AF1 map-based cloning

Utilizes red silk Hui No. 10 and af1 as parent constructsF of construction ₂ Group, total 1200 mutant phenotype plants for gene mapping. At F ₂ 10 normal strains and 10 yellow-green leaf mutant strains are respectively selected from the population and used for constructing a normal gene pool and a mutant gene pool, 400 pairs of SSR primer pairs red silk hui 10 and af1 which are uniformly distributed in the whole genome of rice and stored in a laboratory are selected for carrying out polymorphism analysis, and related primer information is shown in table 1. The results showed that the two primers ZTQ48 and RM3664 located on chromosome 5 were polymorphic between the gene pools, further utilizing F ₂ The 70 recessive individuals in the population are verified, and the two markers are found to be linked with mutation sites, so that the target gene is initially positioned between ZTQ48 and RM3664, and the genetic distances between the target gene and the two markers are 23.86cM and 21.59cM respectively. Within the interval of primary localization, SSR and InDel primers with polymorphisms were further developed, finally locating AF1 between markers InDel4 and InDel5 with a physical distance of 40kb (fig. 4A). According to the gene annotation information provided by the national rice data center (https:// www.ricedata.cn/gene), the interval has 16 annotation genes.

TABLE 1 primer pair information

Sequencing analysis showed that there was a C base to T base substitution in the 3' UTR region of the LOC _ Os05g45030 gene in the af1 mutant (FIG. 4A), the nucleotide sequence of the gene is shown in SEQ ID No.1, the amino acid sequence of the encoded protein is shown in SEQ ID No.2, and the encoded protein is a PSI assembly factor protein by analysis of the amino acid sequence of the encoded protein.

Constructing a complementary vector, transforming af1 mutant callus to obtain a positive transgenic plant, and comparing the positive transgenic plant with the mutant, wherein the color of the leaf of the positive transgenic plant is recovered to be normal green and is the same as that of the wild leaf; the transmission electron microscope result shows that compared with the mutant, the chloroplast of the positive transgenic plant develops normally and is similar to the wild type (fig. 4B and C). Chlorophyll content was measured in wild type, af1 mutant and positive transgenic plants, showing that the chlorophyll content of each leaf of the positive transgenic plants was restored to wild type levels compared to the mutant (fig. 4D). The above experiment confirmed that LOC _ Os05g45030 is the AF1 target gene.

Mutations in the tri, 3' UTR attenuate mRNA stability

The expression level of AF1 in the wild type and the mutant leaf is detected, and the expression level of AF1 in the mutant leaf is found to be significantly lower than that of the wild type (FIG. 5A). The content of AF1 protein in the wild type and the mutant was further analyzed by immunoblotting, and the results showed that the content of AF1 protein in the mutant was significantly decreased compared to the wild type (fig. 5B).

Luciferase experiments were performed with the wild-type and mutant 3 'UTRs transcriptionally fused to the 3' end of Luciferase (LUC). As a result, it was found that the protoplast luciferase activity of rice transformed with the wild-type 3 'UTR was significantly lower than that of the control, while the transcription activity of the mutant 3' -UTR was significantly lower than that of the wild-type (FIG. 5C, D). Further, a normal AF1 cDNA overexpression vector is constructed and is respectively transferred into wild type plants and mutant plants. As a result, compared with the wild type, the color of the leaves of the transgenic plants (AF1OE-WT-1) which are transferred to the normal AF1 cDNA positive genes is slightly deepened, and the chlorophyll content is increased (FIG. 5E); compared with the AF1 mutant, the leaf color of the transgenic plant (AF1OE-AF1-1) positive to the normal AF1 cDNA restored the normal wild-type green phenotype, and the chlorophyll content was similar to the wild-type (FIG. 5F), which indicates that the normal AF1 cDNA can restore the phenotype of the mutant. In conclusion, mutation of the 3' -UTR of the AF1 gene in the mutant reduced the mRNA stability.

Analysis of expression Pattern of AF1

Expression pattern of AF1 was determined by qRT-PCR analysis. AF1 was expressed in all roots, stems, leaves, leaf sheaths, and ears, but the highest expression was observed in leaves (fig. 6A). It was found that when the third leaf appeared completely from the sheath, the shoot contained the fourth to seventh immature leaves. The leaf cells of the upper third (L3U) and lower third (L3L) lobes contain mature chloroplasts, while the Stem Base (SB) and lower fourth (L4) lobes contain mature chloroplasts. The expression level of AF1 in the Stem Base (SB), fourth leaf (L4), second leaf (L2), third leaf lower half leaf (L3L) and third leaf upper half leaf (L3U) was further determined by qRT-PCR analysis, and the results showed that expression was prevalent at these sites, but the highest level was detected in L3L (fig. 6B, C). Since AF1 is a low temperature sensitive mutant, the mutant was exposed to a low temperature of 4 ℃ and then tested for AF1 expression at different times, and the highest expression was found after 6h of treatment (FIG. 6D).

To analyze the expression pattern of AF1 in more detail, longitudinal and transverse AF1 signal expression of Shoot Apical Meristem (SAM) was detected by in situ hybridization technique (fig. 6E). First, a strong signal was observed in the SAM (fig. 6E 1). Next, a uniformly distributed signal was detected in the P2 primordia (FIG. 6E 2). In the P3 primordia, the AF1 signal was concentrated on the edge portions of the vascular bundle protolayer and the leaf primordia (fig. 6E 2). In the P4 primordia, the AF1 signal was mainly distributed in the vascular bundle (fig. 6E3), and both xylem and phloem were expressed (fig. 6E 4). Thus, the expression pattern of AF1 in leaves suggests that AF1 may be involved in leaf development.

Functional analysis of AF1 protein

Species such as maize (Zea mays), tomato (tomato lycopersicum), Arabidopsis thaliana (Arabidopsis thaliana), poplar (Populus trichocarpa), Sorghum (Sorghum bicolor), Brachypodium distachyon (Brachypodium distachyon) were selected among Phytozome, blast-aligned with the AF1 protein sequence, and a phylogenetic tree was constructed using MEGA (fig. 7A). Phylogenetic tree results show that the AF1 gene is widely present in dicotyledonous plants, monocotyledonous plants, mosses and algae plants and shows high homology, which indicates that the AF1 gene in rice is likely to be evolved from algae.

To verify if AF1 was chloroplast localized, the entire CDS of AF1 was fused to the N-terminus of GFP under the drive of the CaMV35S promoter, and constructs were transiently expressed in rice protoplasts (fig. 7B). The OsAF1-GFP fusion protein is indeed localized to chloroplasts. And, AF1 contained the chloroplast transit peptide as predicted by Chlorop. Thus, by truncating the structureAnalysis of chloroplast transit peptide, OsAF1 ^1-38aa And OsAF1 ^39-271aa . The subcellular localization result shows that OsAF1 ^1-38aa Localized in chloroplasts.

Interaction study of AF1 encoding protein and PSI assembly protein

Map-based clone prediction revealed that the protein encoded by AF1 was a PSI assembly protein. To analyze the effects of AF1 with other PSI assembly proteins, the TMpred website (C.) (https://ch.embnet.org/software/TMPRED_form.html) The rice AF1 protein is subjected to transmembrane domain analysis, and the result shows that the AF1 protein is probably embedded on the membrane. Therefore, the interaction analysis of AF1 and other PSI assembly related proteins was performed by using a membrane system yeast double-hybridization experiment. The results indicate that AF1 interacts with PYG7 (fig. 8A). In addition, the interaction of AF1 with PYG7 was further verified by bimolecular fluorescence complementation assay (BiFC) results (fig. 8B). Finally the fact that AF1 interacted with PYG7 was again verified using the Pull-down interaction experiment (fig. 8C). In addition, qRT-PCR analysis found that PYG7 was expressed in all tissues, but was most highly expressed in leaves, with a pattern similar to that of AF1 (fig. 8D). The above experimental results demonstrate that AF1 may play a role in the assembly of PSI through interaction with PYG 7.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather as the intention of all modifications, equivalents, improvements, and equivalents falling within the spirit and scope of the invention.

Sequence listing

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gcgacacagt tgacgcaggc caaacaatgg tctcggttca ttagaattgc aatgtgagtg 1380

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Val Ala Arg Ile Leu Ser Asp Glu Gly Gly Val Gly Gly Asp Met Ile

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Asp Ser Gln Ala Phe Tyr Asp Phe Asn Glu Arg Arg Gly Lys Ala Gly

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Claims

1. The nucleotide sequence of the rice leaf color regulating gene AF1 is shown in SEQ ID No.1, and the amino acid sequence of the coded protein is shown in SEQ ID No. 2.

2. A recombinant expression vector constructed from the rice leaf color regulatory gene AF1 according to claim 1.

3. The rice leaf color regulatory gene AF1 of claim 1.

4. The rice leaf color regulatory gene AF1 of claim 1.

5. The rice leaf color regulatory gene AF1 of claim 1, the recombinant expression vector of claim 2, the reverse sequence of claim 3, and the complementary sequence of claim 4 are used in rice molecular breeding.