Rice white leaf and panicle gene <i>WLP3 </i>and application thereof in rice stress resistance and yield increase

Abstract

A rice white leaf and panicle gene wlp3 is provided. The cDNA sequence of the white leaf and panicle gene wlp3 is shown in SEQ ID NO: 1, and the encoded amino acid sequence of the protein is shown in SEQ ID NO: 2. The rice white leaf and panicle gene wlp3 is applied to rice stress resistance and yield increase. The white leaf and panicle gene wlp3 is configured to improve cold tolerance of plants, enhance photosynthetic rate, increase plant height, leaf albinism at seedling stage, panicle albinism at heading stage, and increase panicle length at low temperature. The present disclosure obtains the rice white leaf and panicle gene wlp3 through screening and mutagenesis, which is related to the stress resistance and chlorophyll synthesis of rice. Therefore, the present disclosure provides a foundation for rice breeding.

Claims

1. A method of expressing cold tolerance genes of rice plants comprising the step of mutating a rice white leaf and panicle gene WLP3, wherein the mutation results in a single base substitution in SEQ ID NO: 3 where nucleotide T at position 380 is changed to C, and the encoded protein has the amino acid sequence of SEQ ID NO: 2, and treating the rice plants to a temperature of 4 C. for 7 days; wherein the rice plants are japonica rice variety Zhonghua 11 (ZH11); and wherein expression levels of genes DREB1A, DREB1B, and DREB2B are increased in the rice plants compared to expression levels of DREB1A, DREB1B, and DREB2B in wild type ZH11 rice plants treated to a temperature of 4 C. for 7 days.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In order to explain the embodiments of the present disclosure or the technical solutions in the prior art more clearly, the following drawings that need to be used in the description of the embodiments or the prior art are briefly introduced. Obviously, the drawings in the following description are only embodiments of the present disclosure. For those of ordinary skill in the art, other drawings can be obtained based on the drawings disclosed without creative work.

(2) FIGS. 1A-1D show the phenotypes of wild type Zhonghua 11 and mutant wlp3; (FIG. 1A) leaf phenotypes of wild type and wlp3 at seedling stage, with the scale of 1 cm; (FIG. 1B) phenotypes of wild type and wlp3 at tillering stage, with the scale of 10 cm; (FIG. 1C) phenotypes of wild type and wlp3 at mature stage, with the scale of 20 cm; (FIG. 1D) panicle phenotypes of wild type and wlp3, with the scale of 5 cm;

(3) FIGS. 2A-2I show the leaf phenotypes of wild type and wlp3 at different temperatures, as well as the chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car) content of the leaf 2 and leaf 3;

(4) FIGS. 3A-3F show the localization map of the wlp3 gene; (FIG. 3A) initial localization of wlp3; (FIGS. 3B-3C) fine localization of WLP3; (FIG. 3D) the gene structure of wlp3; (FIG. 3E) mutation site of wlp3 shown in SEQ ID NO: 79; (FIG. 3F) mutations in the amino-acid sequence of wlp3 shown in SEQ ID NO: 80 and SEQ ID NO: 81;

(5) FIGS. 4A-4E show the response of wild type and wlp3 to cold stress; (FIG. 4A) the phenotypes of wild type and wlp3 before cold treatment at 4 C., with the scale of 5 cm; (FIG. 4B) the expression levels of cold tolerance related genes in wild type and wlp3 before cold treatment at 4 C.; (FIG. 4C) the expression levels of cold tolerance related genes in wild type and wlp3 after cold treatment at 4 C.; (FIG. 4D) the phenotypes of wild type and wlp3 after cold treatment at 4 C., with the scale of 5 cm; (FIG. 4E) the chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoids (Car) content of wild type and wlp3 after cold treatment at 4 C.;

(6) FIGS. 5A-5C show the analysis of expression levels of genes related to leaf color in wild type and wlp3; (FIG. 5A) the expression levels of genes related to chlorophyll synthesis in wild type and wlp3; (FIG. 5B) the expression levels of genes related to ribosome in wild type and wlp3; (FIG. 5C) the expression levels of genes related to chloroplast development in wild type and wlp3;

(7) FIGS. 6A-6B show the functional complementarity diagram of wlp3.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(8) Technical solutions in the embodiments of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the embodiments. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of them. Based on the embodiments of the disclosure, all other embodiments made by those skilled in the art without sparing any creative effort should fall within the protection scope of the disclosure.

Embodiment 1 Acquisition of Mutant Materials

(9) Japonica rice variety Zhonghua 11 was chemically mutagenized by EMS, and the seeds were soaked in water for 8 h. The water was then drained and 1% EMS solution that has been prepared was poured in for soaking to induce mutagenesis for 8 hours. During this period, the mixture was stirred several times with a small wooden stick to ensure uniformity. After the mutagenesis is completed, the toxic EMS buffer solution was washed out with running water. After germination at 25 C., the seedlings were transplanted and harvested. The growth period was managed according to conventional field management. One copy of the white leaf and panicle mutant wlp3 was obtained by screening.

(10) The traits of the mutant have been stably inherited through multiple generations of self-crossing, and all rice materials were planted in the experimental field of College of Biochemistry, Zhejiang Normal University, Jinhua City, Zhejiang Province, under routine management.

Embodiment 2 Phenotype Analysis of the Plants

(11) Under field conditions, the mutant wlp3 showed a white stripe phenotype at the two-leaf stage and lasted until the tillering stage. White stripes were distributed in the whole leaf along the leaf vein. After the four-leaf stage, with the growth and development of rice, the newly grown leaves of wlp3 gradually turned green until they returned to the same phenotype as the wild type. During the growth process, the phenotype of wlp3 was basically the same as that of wild type, while the plant height was higher than that of wild type at mature stage. At the young panicle stage, the panicles of wlp3 showed albinism phenotype again and the panicle length was longer than that of wild type. (FIGS. 1A-1D)

Embodiment 3 Population Construction and Genetic Analysis

(12) When the mutant wlp3 was subjected to reciprocal crosses with conventional indica rice 9311 and ZF802, the F1 plants showed normal wild type phenotype, indicating that wlp3 was controlled by recessive nuclear genes. The segregation ratio of F2 segregating population was counted (Table 1). The results showed that the segregation ratio of normal phenotype plants and mutant phenotype plants was close to 3:1 by chi-square test, which indicated that the white leaf and panicle phenotype of wlp3 was controlled by a pair of single recessive nuclear genes.

(13) TABLE-US-00001 TABLE 1 Genetic analysis of white leaf and panicle mutant wlp3 F2 phenotype Cross Green White combination leaf leaf .sup.2 (/) F1 phenotype and panicle and panicle Total (3:1) wlp3/9311 Normal 332 115 447 0.12 9311/wlp3 phenotype 386 138 524 0.498 wlp3/ZF802 261 81 342 0.316 ZF802/wlp3 189 60 249 0.108

Embodiment 4 Fine Mapping of Wlp3 Gene

(14) Polymorphism screening was conducted on mutants and Zhonghua 11, 9311 by SSR primers uniformly distributed on 12 rice chromosomes preserved in our laboratory. Then, linkage analysis was conducted on 21 F.sub.2 individuals with white leaves and panicles to preliminarily confirm the chromosomal location of the target gene. Genomic DNA was extracted by the CTAB method. The specific steps are as follows:

(15) {circle around (1)} 0.1 g of rice leaves was weighed and grind into powder with liquid nitrogen, then 600 L of DNA extraction buffer prepared by CTAB solution (2% (m/V) CTAB, 100 mmol/L Tris-HCl, 20 mmol/L EDTA, 1.4 mol/L NaCl; pH8.0) was added and subjected to a water bath at 65 C. for 40 min. Then 600 L of chloroform: isoamyl alcohol (volume ratio 24:1) was added and mixed well. Centrifugation was performed at 10,000 rpm for 5 min and the supernatant was transferred to a new centrifuge tube.

(16) {circle around (2)} -1 times the volume of pre-cooled (to 4 C.) isopropanol was added to the supernatant obtained after centrifugation in step {circle around (1)} above, and gently mixed until DNA precipitated. Centrifugation was performed at 13,000 rpm for 8 min and the supernatant was poured out.

(17) {circle around (3)} The DNA precipitate obtained in step {circle around (2)} above was washed with 200 L of 70% (volume concentration) ethanol.

(18) {circle around (4)} The above washed DNA was dried and dissolved in 100 L TE buffer or pure water.

(19) {circle around (5)} The concentration of DNA sample obtained in step {circle around (4)} above was detected by UV spectrophotometry, and the integrity of DNA was detected by 0.7% agarose gel electrophoresis. Complete and appropriate DNA was used for PCR amplification, and incomplete DNA was re-extracted until complete DNA was obtained.

(20) The PCR reaction system was a 10 L system: 1 L of DNA template, 1 L of 10PCR buffer, 0.5 Ml of forward primers (10 mol/L) and 0.5 Ml of reverse primers (10 mol/L), 1 L of dNTPs, 0.2 L of rTaq enzyme, and ddH2O to make up 10 L. The PCR amplification procedure was as follows: pre-denaturation at 94 C. for 4 min; denaturation at 94 C. for 30 s, annealing at 55 C.-60 C. for 30s (temperature varies with primers), extension at 72 C. for 30 s, 40 cycles; and finally, extension at 72 C. for 10 min.

(21) PCR products were electrophoresed on a 4% agarose gel. After electrophoresis, the gel was photographed and read on a gel imager. Linkage analysis of the wlp3 gene using the 120 pairs of SSR primers screened above revealed that linkage was exhibited near the end of chromosome 3. New Indel markers were designed upstream and downstream of the linkage markers, and the 21 individual plants were used to lock the target gene interval between the molecular markers M1 and M6. New molecular markers were designed in this interval again, and 138 F.sub.2 individual plants were used to finally map the gene in the interval between M1 and M2 of about 56 kb. Primer sequences are shown in Table 2.

(22) TABLE-US-00002 TABLE2 Molecularmarkersforfinemapping Primername Forwardprimer(5-3) Reverseprimer(5-3) Purpose B3-22 GTTTTGTTCCTTTGTT AATAGATGAAGGGAGTATC TTCTC(SEQIDNO:5) TCAT(SEQIDNO:6) B3-23 GAGTTACCTCCATCC AGAGTCTTGATCTCGTGCTT Mapping TGTTGC(SEQIDNO:7) C(SEQIDNO:8) M1 CCAGCCTAGTCAGAG GGTGGTTTTGATCCTGGTTT GCAGA(SEQIDNO:9) T(SEQIDNO:10) M2 GAGCTGCCATGGTAG AACGGACAGCTCGTACATT GATGT(SEQIDNO:11) TTT(SEQIDNO:12) M3 GTCTTCCGGTTAGCT TAATTCGCAGCCATTCAAC CCACA(SEQIDNO:13) A(SEQIDNO:14) M4 CAATCGCCTCTACGT GCCGGGATGTTCTGGTAGTA CCATT(SEQIDNO:15) (SEQIDNO:16) M5 GGATTGAAGCAGGTG TGTAGTAGGTGGGGCCGTA TTCGT(SEQIDNO:17) G(SEQIDNO:18) M6 CAAGACTTGGCTGCG CAGTCCGGAAGGAGTATAA TTCTT(SEQIDNO:19) CCA(SEQIDNO:20) M7 GACTTCAATGCGGAA TTGCAGACTCAACCTACAC ACCAT(SEQIDNO:21) CA(SEQIDNO:22) WLP3-1 ATCTCCTCGTGGACA ATGCCACATGCATAAAACC Sequencing TAGGC(SEQIDNO:23) A(SEQIDNO:24) WLP3-2 CTGACGATCCTTTGC AAATGCTCCCAGCTTTTCCT TGATG(SEQIDNO:25) (SEQIDNO:26)

(23) According to the data information of rice genome database (rice.plantbiology.msu.edu), the accession number is LOC_Os03g61620, indicating that this gene is a candidate gene. Primers covering this gene region were used to amplify the DNA of wild type and mutant, respectively. Sequencing results showed that there was a T-to-C mutation in the third exon of WLP3 gene and the coding region 380, resulting in the protein changing from isoleucine to threonine (FIGS. 3A-3F).

(24) The cDNA sequence of rice white leaf and panicle gene wlp3 is described in SEQ ID NO: 1, and the amino acid sequence is described in SEQ ID NO: 2.

(25) The cDNA sequence of WLP3 in Zhonghua 11 is as described in SEQ ID NO: 3, and the amino acid sequence is as described in SEQ ID NO: 4.

(26) The base substitution on the WLP3 gene obtained by the disclosure causes the phenotype of leaf albinism and panicle albinism of rice plants.

Embodiment 5 Wlp3 Response to Cold Stress

(27) Low temperature stress: wild type and wlp3 plants at seedling stage were selected, transferred to the incubator at 4 C. and 28 C. respectively, and treated for 7 days under the same other culture conditions. Subsequently, the chlorophyll content of wild type and wlp3 at different temperatures was measured, and RNA was extracted to analyze the expression levels of genes related to cold stress.

(28) The obtained results are shown in FIGS. 4A-4E. According to FIGS. 4A-4E, it can be seen that after cold treatment, the leaves of wild type turned yellow, while the leaves of wlp3 remained green. The expression levels of DREB1A, DREB1B and DREB2B genes related to cold tolerance were detected by qRT-PCR. It was found that the expression levels of these genes in wlp3 increased sharply after cold treatment, which indicated that the mutation of wlp3 would cause the improvement of plant cold tolerance.

Embodiment 6 Expression Analysis of Genes Related to Chloroplast Development

(29) In order to analyze whether the mutant wlp3 affects the expression of genes related to chlorophyll synthesis and chloroplast development, the disclosure analyzed the expression of genes related to these pathways by qRT-PCR method.

(30) For RNA extraction, total RNA of mutant and wild type leaf samples at tillering stage was isolated according to the steps of total RNA extraction kit RNeasy Plant Mini Kit (QIAGEN), and then reversely transcribed into cDNA according to the instructions of reverse transcription kit ReverTra Ace qPCR RT Kit (TOYOBO). The expression levels of each gene in wild type and mutant were analyzed through quantitative real-time PCR (qRT-PCR). The gene OsActin was used as the internal reference gene, and three parallel duplicate wells were made for each reaction. Relative quantitative analysis was performed through 2.sup.Ct method, and three independent reactions were repeated. The real-time PCR instrument was 7500 real-time PCR system (Applied Biosystems, Life technologies). The obtained experimental data were statistically analyzed through Excel and SPSS19.0 software, and the differences among different data were compared through t-test. The qRT-PCR reaction system (10 L) was: 1 L of cDNA template, 6 L of SYBR qPCR Mix (TOYOBO), 1 L of forward primer (10 mol/L), 1 L of reverse primer (10 mol/L), and ddH2O to make up 10 L. The qRT-PCR amplification procedure was as follows: 95 C. for 30 s; 95 C. for 5 s; 55 C. for 10 s; 72 C. for 15 s, 40 cycles. The required primers are shown in Table 3. The results are shown in FIGS. 5A-5C. The related genes involved in chlorophyll synthesis and chloroplast development showed down-regulated expression in wlp3, indicating that mutations in wlp3 affected chlorophyll synthesis and chloroplast development.

(31) TABLE-US-00003 TABLE3 Primersequencesofreal-time fluorescentquantitativePCR Primer Forward name primer(5-3) Reverseprimer(5-3) qRT-HEMA1 CGCTATTTCTGATGC TCTTGGGTGATGATTGTTTGG TATGGGT (SEQIDNO:28) (SEQIDNO:27) qRT-PORA1 TGTACTGGAGCTGGA GAGCACAGCAAAATCCTAGACG ACAACAA (SEQIDNO:30) (SEQIDNO:29) qRT-CAO1 GATCCATACCCGATC CGAGAGACATCCGGTAGAGC GACAT (SEQIDNO:32) (SEQIDNO:31) qRT-YGL1 AACCTTACCGTCCTA CCATACATCTAACAGAGCACCC TTCCTT (SEQIDNO:34) (SEQIDNO:33) qRT-V1 TGGAGGTCGGGACAG CGAGGAGCACCACCATCAC AGGA (SEQIDNO:36) (SEQIDNO:35) qRT-V2 CGACAAGCAGAGCGA AGGTTGCTGCTCCTTGAATGT AGCG (SEQIDNO:38) (SEQIDNO:37) qRT-rbcS TCCGCTGAGTTTTGG GGACTTGAGCCCTGGAAGG CTATTT (SEQIDNO:40) (SEQIDNO:39) qRT-rbcL CTTGGCAGCATTCCG ACAACGGGCTCGATGTGATA AGTAA (SEQIDNO:42) (SEQIDNO:41) qRT-Cab1R AGATGGGTTTAGTGC TTTGGGATCGAGGGAGTATTT GACGAG (SEQIDNO:44) (SEQIDNO:43) qRT-Cab2R TGTTCTCCATGTTCG GCTACGGTCCCCACTTCACT GCTTCT (SEQIDNO:46) (SEQIDNO:45) qRT-psaA GCGAGCAAATAAAAC GTACCAGCTTAACGTGGGGAG ACCTTTC (SEQIDNO:48) (SEQIDNO:47) qRT-psbA CCCTCATTAGCAGAT ATGATTGTATTCCAGGCAGAGC TCGTTTT (SEQIDNO:50) (SEQIDNO:49) qRT-spp CGGAGAGGAAACATA ATAGGCATTTGTCTTTGTCTC ATGAC (SEQIDNO:52) (SEQIDNO:51) qRT- CTAAGACCGAATGAC GCACTGCCAACAAGAATACC OsPPR1 AAATGC (SEQIDNO:54) (SEQIDNO:53) qRT-OsDVR CGAGCCCAGGTTCAT CCTCCCGATCTTGCCGAACTCC CAAGGTGC (SEQIDNO:56) (SEQIDNO:55) qRT-rpoA GTGGAAGTGTGTTGA TCTCTCTTGATCCGTAACTC ATCAA (SEQIDNO:58) (SEQIDNO:57) qRT-rpoB TTTGGTTTCGATGTG TATGGTCTAATTCCGAGCGGT CA (SEQIDNO:60) (SEQIDNO:59) qRT- AAGCAGACAGTGATG ATCACATGCATGCACCCAAA OsRpoTp ACATC (SEQIDNO:62) (SEQIDNO:61) qRT-rps12 AGCCGTTTGCTACCA TGATCGGTACCAATGAATAGG ATGG (SEQIDNO:64) (SEQIDNO:63) qRT-WLP1 TTGATGACTATTTGA ACATCAAGACGACCCACAGTAA AGGGTTGG (SEQIDNO:66) (SEQIDNO:65) qRT-WGL2 GCCAAGGAGTATTTG TAACTTTGTTTGCGGTGCTG CAAGG (SEQIDNO:68) (SEQIDNO:67) qRT-ASL4 ACTGCTTTCTTGCCT GAAGCTGTCTGCACCTTTCC TTGGA (SEQIDNO:70) (SEQIDNO:69) qRT-WLP3 GGAGAGGCCAAGACT CAAGCAAGACTTGGCAATCA CAGTG (SEQIDNO:72) (SEQIDNO:71) qRT-ASL1 AAGAAAGCTGATGCC ACCAACCACGGAGTATCTCG ACACC (SEQIDNO:74) (SEQIDNO:73) qRT-ASL2 CTGCTGTTCATGCAG CAGGGAAGTCCTCGTATCCA TGGTT (SEQIDNO:76) (SEQIDNO:75) qRT-AL2 AGAAGACGGAGTTCG TGACACCCTCCTTGACCTTC ACGTG (SEQIDNO:78) (SEQIDNO:77)

Embodiment 7 Transgenic Experiment

(32) The transgenic experiment was performed on the genes mentioned in SEQ ID NO: 1. The 700 bp promoter upstream of wlp3 and the DNA of entire genome of wlp3 were amplified by the primer pairs respectively for the construction of complementary vectors, and were transferred into the healing tissue of wlp3 by Agrobacterium. The obtained result is that a total of 21 transgenic plants were obtained after transformation. Observing the phenotypes, it was found that 2 plants still exhibited an albinism phenotype during the seedling stage, while 19 transgenic plants restored their green phenotype. Through identification, it was found that the 19 transgenic plants were positive and all exhibited normal phenotypes. The genetic complementarity of wlp3 confirmed that LOC_Os03g61260 is the WLP3 gene, and the single base mutation of this gene can lead to the appearance of albinism phenotype in rice seedlings and later turn green (as shown in FIGS. 6A-6B).

(33) The agronomic traits of wild type and wlp3 mutant are compared in Table 4;

(34) TABLE-US-00004 TABLE 4 Agronomic traits of wild type and wlp3 Trait ZH11 wlp3 Plant height/cm 106.20 2.17 129.73 6.64** Panicle length/cm 20.32 1.32 26.90 1.97** Effective number of panicles 10.60 3.21 8.40 1.52 Flag leaf length/cm 36.00 8.19 26.93 2.54 Primary branch number 12.00 1.00 14.40 1.52* Secondary branch number 23.80 6.46 33.00 2.65* Tiller number 9.80 2.39 9.20 1.30 Filled grain number per panicle 121.00 23.97 150.00 11.00** Seed-setting rate/% 41.65 1.74 48.75 2.20** 1000-grain weight/g 18.90 0.46 15.90 0.78*

(35) Various embodiments in the present specification are described in a progressive manner, and the emphasizing description of each embodiment is different from the other embodiments. The same and similar parts of various embodiments can be referred to for each other.

(36) The above description of the disclosed embodiments enables those skilled in the art to realize or use the present disclosure. Many modifications to these embodiments will be apparent to those skilled in the art. The general principle defined herein can be realized in other embodiments without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principle and novel features disclosed herein.