水稻矮化劍葉卷曲基因DCFL1的圖位克隆和功能研究
發(fā)布時間：2019-06-21 21:18
【摘要】：株高是水稻最重要的農(nóng)藝性狀之一,直接影響水稻的抗倒伏和產(chǎn)量,大量矮桿突變體的出現(xiàn)引發(fā)了“第二次綠色革命”。劍葉是水稻最重要的功能葉,其形態(tài)特征關(guān)系到葉片間的通透性和光能利用率,直接影響水稻灌漿速度和籽粒飽滿度。因此,了解水稻株高的遺傳模式和劍葉發(fā)育的分子機(jī)理對水稻株型改良和豐產(chǎn)具有極其重要的意義。利用EMS誘變秈型水稻恢復(fù)系縉恢10號,從其后代鑒定到一個矮化劍葉基部特異卷曲突變體,暫命名為dcfl1(dwarf and curled flag leaf 1)。本文對dcfl1進(jìn)行了表型和細(xì)胞學(xué)觀察,光合色素含量和光合參數(shù)分析,并開展了基因定位、圖位克隆、表達(dá)模式分析和功能驗(yàn)證等研究。主要結(jié)果如下:1.dcfl1的表型分析和農(nóng)藝性狀調(diào)查突變體dcfl1全生育期表現(xiàn)出矮化性狀,dcfl1的細(xì)胞長度明顯比野生型短,達(dá)到了極顯著水平,而細(xì)胞寬度無顯著差異。抽穗期dcfl1劍葉的葉片和葉鞘連接處硬化,劍葉基部展開受阻,半邊葉片向內(nèi)卷曲。劍葉上部和中部正常,其他葉片也正常。農(nóng)藝性狀分析發(fā)現(xiàn),與野生型相比,dcfl1的有效穗數(shù)為14.2,極顯著高于野生型的11.6,穗粒數(shù)、實(shí)粒數(shù)、結(jié)實(shí)率和千粒重等則無顯著變化。2.dcfl1的光合色素含量和光合參數(shù)測定開花期對光合色素含量測定結(jié)果顯示,dcfl1的劍葉、倒2葉和倒3葉的葉綠素a含量均極顯著高于野生型,類胡蘿卜素含量也略有升高,但僅劍葉達(dá)到極顯著差異水平,葉綠素b含量則無顯著變化。光合參數(shù)測定發(fā)現(xiàn),與野生型相比,dcfl1的胞間CO2濃度無明顯變化,凈光合速率Pn、氣孔導(dǎo)度Gs和蒸騰速率Tr則顯著降低。3.dcfl1的遺傳分析與基因定位利用雜交組合西農(nóng)1A/dcfl1的F1、F2群體進(jìn)行遺傳分析,表明dcfl1突變表型受1對隱性核基因控制。以西農(nóng)1A/dcfl1雜交組合的620株F2隱性單株為定位群體,利用SSR標(biāo)記和In Del標(biāo)記進(jìn)行基因定位,最終將DCFL1定位在第3染色體Ind03-11和Ind03-6之間,物理距離約為78kb。4.DCFL1基因的克隆與蛋白分析對定位區(qū)間內(nèi)的15個注釋基因進(jìn)行基因測序,發(fā)現(xiàn)LOC_Os03g04680在dcfl1中發(fā)生了一個G-A的堿基替換,導(dǎo)致編碼的氨基酸從半胱氨酸變成了酪氨酸,初步確定為DCFL1的候選基因。DCFL1編碼細(xì)胞色素P450加單氧酶Os CYP96B4,屬于CYP86家族中的CYP96亞家族。5.DCFL1候選基因的確定利用野生型DCFL1基因組片段包括上游2875bp、編碼框和下游1315bp構(gòu)建互補(bǔ)表達(dá)載體,通過農(nóng)桿菌介導(dǎo)遺傳轉(zhuǎn)化突變體dcfl1。在轉(zhuǎn)基因植株中,我們共鑒定了12株陽性轉(zhuǎn)基因植株,表型觀察發(fā)現(xiàn)突變體表型得以恢復(fù),確定了LOC_Os03g04680是DCFL1基因。6.DCFL1基因的表達(dá)模式分析通過半定量RT-PCR和q PCR等技術(shù)進(jìn)行表達(dá)模式分析,結(jié)果表明DCFL1在根、莖、葉和穗中都有表達(dá);在葉片和葉鞘中表達(dá)量較高,其中,劍葉葉鞘表達(dá)量最高。利用GUS試驗(yàn)進(jìn)一步探究DCFL1基因表達(dá)模式。GUS染色結(jié)果表明DCFL1基因在各器官均有表達(dá),這與半定量RT-PCR和q PCR結(jié)果一致。7.DCFL1基因與激素的關(guān)系激素處理實(shí)驗(yàn)發(fā)現(xiàn)不同濃度的BR和GA3均無法使突變體dcfl1和等位突變體dcfl1-1矮化表型得以恢復(fù)。說明兩個突變體的矮化表型不受外源BR和GA3的影響,但在沒有驗(yàn)證其他植物激素的響應(yīng)以及沒有檢測突變體dcfl1和突變體dcfl1-1內(nèi)源激素的前提下,不能排除DCFL1參與植物激素代謝途徑的可能性。8.DCFL1基因可能參與表皮蠟質(zhì)的合成對突變體dcfl1卷曲的劍葉基部表皮進(jìn)行掃描電鏡觀察發(fā)現(xiàn)dcfl1的兩個小葉脈之間存在溝壑,導(dǎo)致dcfl1表皮蠟質(zhì)覆蓋面積減少。葉綠素浸析試驗(yàn)結(jié)果發(fā)現(xiàn)dcfl1的表皮滲透性升高,導(dǎo)致葉綠素比野生型更容易提取出來,暗示著dcfl1表皮蠟質(zhì)含量要低于野生型。這些結(jié)果表明DCFL1可能參與表皮蠟質(zhì)的合成。
[Abstract]:The plant height is one of the most important agronomic characters of rice, which directly affects the lodging and yield of rice, and the emergence of a large number of short-stem mutants has raised the "the second green revolution". Swordleaf is one of the most important functional leaves of rice, and its morphological characteristics are related to the permeability and light energy utilization of the leaves, which directly affects the grain filling speed and the fullness of the grain. Therefore, it is of great significance to understand the high genetic pattern of rice plant and the molecular mechanism of the development of swordleaf. A dwarf sword leaf base specific curl mutant was identified from the progeny of the rice restorer line of the rice-type rice restorer line of the EMS by using the EMS, and it was temporarily named dcfl1 (dwarf and curled flag leaf 1). In this paper, the phenotype and cytological observation of dcfl1, the content of photosynthetic pigment and the analysis of the photosynthetic parameters were carried out, and the research of gene location, site cloning, expression pattern analysis and functional verification was carried out. The main results are as follows:1. The phenotype and agronomic characters of dcfl1 show that the whole growth period of the mutant dcfl1 shows the dwarfing character, and the cell length of dcfl1 is significantly shorter than that of the wild type, and the cell width is not significantly different. At the junction of the blade and the blade of the dcfl1 swordleaf of the heading stage, the base of the blade was blocked, and the half of the blade was curled inward. The upper part and the middle part of the blade are normal, and the other blades are normal. The results showed that, compared with wild-type, the effective spikelet number of dcfl1 was 14.2, which was significantly higher than that of the wild-type 11.6, the number of kernels per spike, the number of grains per ear, the seed setting and the 1000-grain weight were not changed significantly. The content of chlorophyll a was significantly higher than that of wild type, but the content of carotenoid increased slightly, but the content of chlorophyll b did not change significantly. The results showed that there was no significant change in the intercell CO2 concentration of dcfl1, the net photosynthetic rate (Pn), the stomatal conductance (Gs) and the transpiration rate (Tr) decreased significantly in comparison with the wild type. It is shown that the dcfl1 mutant phenotype is controlled by 1 recessive nuclear gene. The 620 strain F2 recessive single plant of the hybrid combination of the No. 1A/ dcfl1 west of the west is a positioning group, and the gene is positioned by using the SSR marker and the In Del marker, and the DCFL1 is finally positioned between the third chromosome In03-11 and the In03-6, The physical distance was about 78 kb.4. The cloning and protein analysis of the DCFL1 gene were used to sequence the 15 annotation genes in the localization interval, and found that LOC _ Os03g04680 had a G-A base substitution in dcfl1, resulting in the encoded amino acid being transformed from cysteine into tyrosine and preliminarily determined as the candidate gene of DCFL1. DCFL1 encodes a cytochrome P450-derived CYP96B4, which belongs to the CYP96 subfamily of the CYP86 family. In the transgenic plant, we identified 12 positive transgenic plants and found that the phenotype of the mutant was restored. The expression pattern of the DCFL1 gene was analyzed by semi-quantitative RT-PCR and q-PCR. The results showed that DCFL1 was in the root and the stem. The expression of leaf and spikelet was high, and the expression of leaf and leaf was the highest. The expression pattern of DCFL1 gene was further explored by GUS test. The results of GUS staining showed that the DCFL1 gene was expressed in all organs, which was consistent with the results of semi-quantitative RT-PCR and q-PCR. The dwarfing phenotype of the two mutants is not affected by the foreign BR and the GA3, but on the premise of not verifying the response of other plant hormones and the absence of the endogenous hormones of the mutant dcfl1 and the mutant dcfl1-1, The possibility of DCFL1 in the pathway of plant hormone metabolism could not be ruled out. The results of the chlorophyll-leaching test showed that the increase of the skin permeability of dcfl1 resulted in a much easier extraction of the chlorophyll than the wild-type, suggesting that the waxy content of the dcfl1 was lower than that of the wild-type. These results suggest that DCFL1 may be involved in the synthesis of the skin wax.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S511
本文編號：2504359
[Abstract]:The plant height is one of the most important agronomic characters of rice, which directly affects the lodging and yield of rice, and the emergence of a large number of short-stem mutants has raised the "the second green revolution". Swordleaf is one of the most important functional leaves of rice, and its morphological characteristics are related to the permeability and light energy utilization of the leaves, which directly affects the grain filling speed and the fullness of the grain. Therefore, it is of great significance to understand the high genetic pattern of rice plant and the molecular mechanism of the development of swordleaf. A dwarf sword leaf base specific curl mutant was identified from the progeny of the rice restorer line of the rice-type rice restorer line of the EMS by using the EMS, and it was temporarily named dcfl1 (dwarf and curled flag leaf 1). In this paper, the phenotype and cytological observation of dcfl1, the content of photosynthetic pigment and the analysis of the photosynthetic parameters were carried out, and the research of gene location, site cloning, expression pattern analysis and functional verification was carried out. The main results are as follows:1. The phenotype and agronomic characters of dcfl1 show that the whole growth period of the mutant dcfl1 shows the dwarfing character, and the cell length of dcfl1 is significantly shorter than that of the wild type, and the cell width is not significantly different. At the junction of the blade and the blade of the dcfl1 swordleaf of the heading stage, the base of the blade was blocked, and the half of the blade was curled inward. The upper part and the middle part of the blade are normal, and the other blades are normal. The results showed that, compared with wild-type, the effective spikelet number of dcfl1 was 14.2, which was significantly higher than that of the wild-type 11.6, the number of kernels per spike, the number of grains per ear, the seed setting and the 1000-grain weight were not changed significantly. The content of chlorophyll a was significantly higher than that of wild type, but the content of carotenoid increased slightly, but the content of chlorophyll b did not change significantly. The results showed that there was no significant change in the intercell CO2 concentration of dcfl1, the net photosynthetic rate (Pn), the stomatal conductance (Gs) and the transpiration rate (Tr) decreased significantly in comparison with the wild type. It is shown that the dcfl1 mutant phenotype is controlled by 1 recessive nuclear gene. The 620 strain F2 recessive single plant of the hybrid combination of the No. 1A/ dcfl1 west of the west is a positioning group, and the gene is positioned by using the SSR marker and the In Del marker, and the DCFL1 is finally positioned between the third chromosome In03-11 and the In03-6, The physical distance was about 78 kb.4. The cloning and protein analysis of the DCFL1 gene were used to sequence the 15 annotation genes in the localization interval, and found that LOC _ Os03g04680 had a G-A base substitution in dcfl1, resulting in the encoded amino acid being transformed from cysteine into tyrosine and preliminarily determined as the candidate gene of DCFL1. DCFL1 encodes a cytochrome P450-derived CYP96B4, which belongs to the CYP96 subfamily of the CYP86 family. In the transgenic plant, we identified 12 positive transgenic plants and found that the phenotype of the mutant was restored. The expression pattern of the DCFL1 gene was analyzed by semi-quantitative RT-PCR and q-PCR. The results showed that DCFL1 was in the root and the stem. The expression of leaf and spikelet was high, and the expression of leaf and leaf was the highest. The expression pattern of DCFL1 gene was further explored by GUS test. The results of GUS staining showed that the DCFL1 gene was expressed in all organs, which was consistent with the results of semi-quantitative RT-PCR and q-PCR. The dwarfing phenotype of the two mutants is not affected by the foreign BR and the GA3, but on the premise of not verifying the response of other plant hormones and the absence of the endogenous hormones of the mutant dcfl1 and the mutant dcfl1-1, The possibility of DCFL1 in the pathway of plant hormone metabolism could not be ruled out. The results of the chlorophyll-leaching test showed that the increase of the skin permeability of dcfl1 resulted in a much easier extraction of the chlorophyll than the wild-type, suggesting that the waxy content of the dcfl1 was lower than that of the wild-type. These results suggest that DCFL1 may be involved in the synthesis of the skin wax.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S511
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