CN103233024B - Paddy rice lateral root density-related coding gene and use thereof - Google Patents
Paddy rice lateral root density-related coding gene and use thereof Download PDFInfo
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 44
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- 235000009566 rice Nutrition 0.000 title claims abstract description 19
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- 241000209094 Oryza Species 0.000 claims abstract description 18
- 238000009395 breeding Methods 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims 2
- 108700028369 Alleles Proteins 0.000 abstract description 6
- 101150059170 NCED gene Proteins 0.000 abstract description 3
- 238000000034 method Methods 0.000 description 6
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010080953 9-cis-epoxycarotenoid dioxygenase Proteins 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
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- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 230000009105 vegetative growth Effects 0.000 description 2
- NLZUEZXRPGMBCV-UHFFFAOYSA-N Butylhydroxytoluene Chemical compound CC1=CC(C(C)(C)C)=C(O)C(C(C)(C)C)=C1 NLZUEZXRPGMBCV-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 101100079509 Oncidium hybrid cultivar NCED gene Proteins 0.000 description 1
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- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
技术领域technical field
本发明属于分子生物学和农业领域，具体而言，涉及一种水稻侧根密度相关的编码基因及其应用。The invention belongs to the fields of molecular biology and agriculture, and in particular relates to a coding gene related to rice lateral root density and application thereof.
背景技术Background technique
农作物的人工选择一般经历了驯化和改良2个过程，驯化过程一般是将野生种变得容易种植和收获，而改良过程则更加注重作物的经济性状，如产量、品质、抗逆性、适应性等。发掘并克隆作物中与改良有关的基因具有重要的意义，将为我们改良农作物提供重要的基因资源。The artificial selection of crops generally undergoes two processes of domestication and improvement. The domestication process generally makes wild species easier to plant and harvest, while the improvement process pays more attention to the economic traits of crops, such as yield, quality, stress resistance, and adaptability. wait. It is of great significance to discover and clone genes related to improvement in crops, which will provide us with important genetic resources for improving crops.
一直以来，干旱问题一直是影响农作物产量的主要因素之一。目前，应对作物特别是水稻的抗旱问题，绝大部分的研究是基于水稻的自然群体，通过过表达一些转录因子、蛋白激酶等基因，达到相应的抗旱的目的。Drought has always been one of the main factors affecting crop yields. At present, to deal with the drought resistance of crops, especially rice, most of the research is based on the natural population of rice, through the overexpression of some transcription factors, protein kinases and other genes to achieve the corresponding purpose of drought resistance.
发明内容Contents of the invention
本发明提供了一种水稻侧根密度相关的编码基因及其应用，该基因能够提高水稻侧根密度。为了实现本发明的目的，拟采用如下技术方案：The invention provides a coding gene related to the lateral root density of rice and application thereof, and the gene can increase the lateral root density of rice. In order to realize the purpose of the present invention, it is planned to adopt the following technical solutions:
9-顺式-环氧类胡萝卜素双加氧酶(Nced)的基因编号为Os12g0435200(其基因序列为SEQ ID NO.:C)，野生型的基因在1009位的碱基为G而不是A，因此将野生型的NCED基因命名为C型(互补碱基为G)，本发明通过将1009的碱基G替换为A形成具有抗旱作用的等位基因，其基因序列为SEQ ID NO.：1，并将其命名为T型，该等位基因造成基因编码酶的氨基酸从缬氨酸变为异亮氨酸。The gene number of 9-cis-epoxy carotenoid dioxygenase (Nced) is Os12g0435200 (its gene sequence is SEQ ID NO.: C), and the base of the wild-type gene at position 1009 is G instead of A , so the wild-type NCED gene is named C-type (complementary base is G), the present invention forms an allele with drought resistance by replacing the base G of 1009 with A, and its gene sequence is SEQ ID NO.: 1, and named it T-type, this allele causes the amino acid of the gene-encoded enzyme to change from valine to isoleucine.
本发明一方面涉及一种编码基因，其包含SEQ ID NO.:1的基因序列或其互补的基因序列。One aspect of the present invention relates to a coding gene, which comprises the gene sequence of SEQ ID NO.: 1 or its complementary gene sequence.
在本发明的另一方面，本发明还涉及上述由上述编码基因所表达的蛋白。In another aspect of the present invention, the present invention also relates to the above-mentioned protein expressed by the above-mentioned coding gene.
本发明另一方面还涉及含有上述编码基因的载体或者水稻在育种中的应用。Another aspect of the present invention also relates to the vector containing the above-mentioned coding gene or the application of rice in breeding.
本发明还涉及含有上述编码基因的载体在提高水稻侧根密度中的应用，优选的，还包括在提高水稻抗旱能力中的作用。The present invention also relates to the application of the carrier containing the above coding gene in increasing the lateral root density of rice, preferably, it also includes the effect in improving the drought resistance ability of rice.
本发明的Nced基因的T型等位基因能够显著增加陆稻内源的ABA含量，并且使侧根的数目增多，从而使陆稻更加抗旱，从而可以提高在干旱胁迫下陆稻的产量。The T-type allele of the Nced gene of the present invention can significantly increase the endogenous ABA content of upland rice and increase the number of lateral roots, thereby making the upland rice more drought-resistant and increasing the yield of the upland rice under drought stress.
附图说明Description of drawings
图1：2种基因型陆稻的ABA含量；Figure 1: ABA content of two genotypes of upland rice;
图2：F7重组自交系2种基因型家系ABA含量；Figure 2: ABA content of two genotype families of the F7 recombinant inbred line;
图3：2种基因型陆稻的侧根密度；Figure 3: Lateral root density of two genotypes of upland rice;
图4：2种基因型陆稻体视镜下的根系照片；Figure 4: Photos of root systems of two genotypes of upland rice under a stereoscope;
图5：F7重组自交系2种基因型家系的侧根密度；Figure 5: The lateral root density of the two genotype families of the F7 recombinant inbred line;
图6：F7重组自交系2种基因型体视镜下的根系照片。Figure 6: Photos of root systems of two genotypes of the F7 recombinant inbred line under a stereoscope.
具体实施方式：Detailed ways:
实施例1Example 1
酶联免疫(ELISA)法测量ABA：Measurement of ABA by enzyme-linked immunosorbent assay (ELISA):
在温室的控水环境中种植了T型和C型基因型的陆稻(C型和T型均是自然群体中的基因)在陆稻分蘖后的营养生长期间(此时Nced基因是高表达的)，采集20个T型陆稻和17个C型陆稻的叶片(每个0.5g)，使用酶联免疫法进行激素ABA含量的测定。所有的样品在液氮中磨成粉末，用80％的甲醇(含1mmol/L的2,6-二叔丁基-4-甲基苯酚)过夜萃取。将混合液6000rpm离心15分钟，将上清使用氮气吹干，然后溶入磷酸盐缓冲液(pH＝7.5,1％Tween-20,1％glutin)。测量如图1所示，T型陆稻的ABA含量A显著高于C型陆稻的ABA含量(t-test,P＝0.033)。During the vegetative growth period of upland rice after tillering (when Nced gene is highly expressed ), collected 20 T-type upland rice and 17 C-type upland rice leaves (0.5 g each), and used enzyme-linked immunosorbent assay to measure the content of the hormone ABA. All samples were pulverized in liquid nitrogen and extracted with 80% methanol (containing 1 mmol/L 2,6-di-tert-butyl-4-methylphenol) overnight. The mixture was centrifuged at 6000 rpm for 15 minutes, the supernatant was blown dry with nitrogen, and then dissolved in phosphate buffer (pH=7.5, 1% Tween-20, 1% glutin). As shown in Figure 1, the ABA content A of T-type upland rice was significantly higher than that of C-type upland rice (t-test, P=0.033).
另外，使用C型水稻IR64与T型陆稻IRAT104构建了F7世代的重组自交系分离群，此分离群包括11个T型家系与12个C型家系。分别测定了2种家系的ABA含量，如图2所示，T型家系的ABA含量要显著高于C型家系的ABA含量(t-test,P＝0.016)。In addition, the C-type rice IR64 and T-type upland rice IRAT104 were used to construct the recombinant inbred line isolates of the F7 generation, which included 11 T-type families and 12 C-type families. The ABA content of the two families was measured respectively, as shown in Figure 2, the ABA content of the T-type family was significantly higher than that of the C-type family (t-test, P=0.016).
通过以上2个实验，能够得出这样的结论：NCED的T型的等位基因与野生型的C型相比，能够显著提高9-顺式-环氧类胡萝卜素双加氧酶的活性，从而使激素ABA的含量增加。Through the above two experiments, it can be concluded that the T-type allele of NCED can significantly increase the activity of 9-cis-epoxycarotenoid dioxygenase compared with the wild-type C-type, Thereby increasing the content of the hormone ABA.
2.不同基因型的陆稻品系及重组自交系品系的侧根的定量2. Quantification of lateral roots of different genotypes of upland rice lines and recombinant inbred lines
在温室模拟陆稻生长环境的条件下，种植了9种C型陆稻和8种T型陆稻品系。生长至分蘖后的营养生长时期时，将其从土中取出并将根清洗干净(此过程要尽量避免伤害到根系系统)。测量了所有的主根长度，统计了主根上长于1cm的侧根的数目，并计算了侧根的密度(每个植株的侧根总数目/每个植株的主根总长度)。每个品系我们各统计了5个单株并计算了平均值。我们发现，T型陆稻单位长度主根上的侧根平均数要显著高于C型陆稻的数目(t-test,P＝0.009,如图3所示)。图4显示的是C型陆稻与T型陆稻的根系在体视镜下的图片。同时，我们使用同样的方法对对IR64与IRAT104构建的F7代重组自交系不同基因型的家系进行了根系表型的测定，如图5所示，T型家系单位主根上侧根的平均数目要显著高于C型家系的(t-test,P＝0.035)。图6展示的是2种基因型家系的根的表型。Nine C-type upland rice lines and eight T-type upland rice lines were planted in the greenhouse under the condition of simulated upland rice growth environment. When growing to the vegetative growth period after tillering, take it out from the soil and clean the roots (this process should try to avoid damaging the root system). All tap root lengths were measured, the number of lateral roots longer than 1 cm on the tap root was counted, and the lateral root density (total number of lateral roots per plant/total length of main root per plant) was calculated. We counted 5 individual plants for each line and calculated the average value. We found that the average number of lateral roots per unit length of T-type upland rice was significantly higher than that of C-type upland rice (t-test, P=0.009, as shown in Figure 3). Figure 4 shows the pictures of the root systems of C-type upland rice and T-type upland rice under a stereoscope. At the same time, we used the same method to measure the root phenotypes of the families of different genotypes of the F7 generation recombinant inbred lines constructed by IR64 and IRAT104. As shown in Figure 5, the average number of lateral roots on the main root of the T-type family unit is Significantly higher than that of type C family (t-test, P=0.035). Figure 6 shows the root phenotypes of the two genotype families.
通过以上的根系表型测定试验，可以这样的结论：T型陆稻的侧根密度要显著高于C型陆稻的侧根密度。Through the above root phenotype determination experiments, it can be concluded that the lateral root density of T-type upland rice is significantly higher than that of C-type upland rice.
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何不经过创造性劳动想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应该以权利要求书所限定的保护范围为准。The above is only a specific implementation of the present invention, but the scope of protection of the present invention is not limited thereto, and any changes or replacements that do not come to mind through creative work shall be covered within the scope of protection of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope defined in the claims.
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| CN102268081A (en) * | 2011-07-20 | 2011-12-07 | 浙江大学 | Rice lateral root control gene OsIAAII and use thereof |
| CN102584970A (en) * | 2012-02-13 | 2012-07-18 | 浙江大学 | Rice lateral root formation regulatory gene OsLRD1 and protein coded by same |
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| CN102268081A (en) * | 2011-07-20 | 2011-12-07 | 浙江大学 | Rice lateral root control gene OsIAAII and use thereof |
| CN102584970A (en) * | 2012-02-13 | 2012-07-18 | 浙江大学 | Rice lateral root formation regulatory gene OsLRD1 and protein coded by same |
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| Dian.W.M.."GenBank Accession No:AY838898.1".《GenBank》.2004,第1-2页. * |
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