近日，華中農(nóng)業(yè)大學(xué)水產(chǎn)學(xué)院高澤霞教授課題組在Aquaculture期刊上在線發(fā)表了題為“Generation of Megalobrama amblycephala without intermuscular bones by runx2b gene mutation”的研究論文。該研究獲得了肌間刺完全缺失且生長(zhǎng)發(fā)育正常的團(tuán)頭魴F1代突變個(gè)體，不僅為獲得可穩(wěn)定遺傳的團(tuán)頭魴新品種奠定了重要基礎(chǔ)，也為無(wú)肌間刺性狀從模式物種到經(jīng)濟(jì)魚類上的應(yīng)用提供了很好的示例。

 

　　肌間刺起源于肌腱祖細(xì)胞，膜內(nèi)骨化而來(lái)，僅存在于低等真骨魚類中。世界主要養(yǎng)殖魚類都含有一定數(shù)量的肌間刺，特別是我國(guó)主養(yǎng)的青草鰱鳙鯉鯽魴等大宗淡水魚類，食用時(shí)極易出現(xiàn)卡喉嚨的危險(xiǎn)，嚴(yán)重影響了其水產(chǎn)品的經(jīng)濟(jì)價(jià)值。課題組前期以模式魚類斑馬魚為研究材料，篩選鑒定出調(diào)控魚類肌間刺發(fā)生的關(guān)鍵基因runx2b，2022年發(fā)表于National Science Reivew上。接著，課題組開始探索是否可以將這一模式物種上的研究發(fā)現(xiàn)應(yīng)用到經(jīng)濟(jì)魚類上。

 

　　課題組以長(zhǎng)期開展遺傳選育工作的團(tuán)頭魴（俗稱武昌魚）為經(jīng)濟(jì)養(yǎng)殖魚類代表開展驗(yàn)證工作。團(tuán)頭魴屬于鯉形目、鯉科、鲌亞科、魴屬魚類，是我國(guó)重要的淡水養(yǎng)殖經(jīng)濟(jì)魚類，約含118根肌間刺。課題組前期已通過(guò)分子輔助選育技術(shù)培育出生長(zhǎng)速度快、成活率高的團(tuán)頭魴“華海1號(hào)”國(guó)審新品種，在該品種的基礎(chǔ)上，采用基因編輯獲得肌間刺部分缺失的團(tuán)頭魴突變體F0代（肌間刺缺失5.6%–33.8%），進(jìn)一步將F0代雌雄突變個(gè)體進(jìn)行人工繁殖獲得F1代。課題組隨機(jī)篩選45天的團(tuán)頭魴F1代品系60條，運(yùn)用茜素紅整體骨骼染色方法觀察團(tuán)頭魴F1代肌間刺的表型，結(jié)果顯示27條團(tuán)頭魴的肌間刺完全缺失。在F1代群體4月齡時(shí)，課題組利用便攜式X光掃描儀在5000尾個(gè)體中篩選到了316尾完全沒(méi)有肌間刺的團(tuán)頭魴個(gè)體，可作為2023年繁殖無(wú)肌間刺團(tuán)頭魴的親本。同時(shí)，研究發(fā)現(xiàn)完全無(wú)肌間刺團(tuán)頭魴F1代幼魚個(gè)體骨骼的礦化、肌肉脂肪酸和氨基酸含量等性狀均未受到顯著影響。目前，無(wú)肌間刺團(tuán)頭魴生長(zhǎng)性狀良好，6月齡無(wú)刺團(tuán)頭魴平均體重達(dá)到了150g以上，部分個(gè)體已達(dá)到250 g。課題組后期將進(jìn)一步跟蹤研究無(wú)肌間刺團(tuán)頭魴成魚的骨骼礦化、肌肉營(yíng)養(yǎng)成分等性狀差異情況。

 

　　華中農(nóng)業(yè)大學(xué)水產(chǎn)學(xué)院碩士生董強(qiáng)和博士后聶春紅為論文共同第一作者，水產(chǎn)學(xué)院高澤霞教授為論文通訊作者。該研究得到國(guó)家大宗淡水魚類產(chǎn)業(yè)技術(shù)體系、湖北洪山實(shí)驗(yàn)室、國(guó)家自然科學(xué)基金、國(guó)家重點(diǎn)研發(fā)計(jì)劃等項(xiàng)目的支持。

 

　　【英文摘要】

 

　　Blunt snout bream （Megalobrama amblycephala） is an important herbivorous freshwater economic fish cultivated in China. Its economic value has been affected by the existence of intermuscular bones （IBs）。 Our previous study proved that runx2b is the key gene for the IBs formation, and lack of runx2b gene could obtain a stable zebrafish mutant without IBs. We found runx2b mutant F0 generation of blunt snout bream through CRISPR/Cas9 editing technology had a significant reduction on the number of IBs. In order to further obtain the blunt snout bream without IBs, we used the runx2b mutant F0 generation to self-bred to obtain F1 generation. Subsequently, the 35 runx2b mutant F1 individuals of blunt snout bream without IBs were genotyped and found the number of runx2b targeting sites 1, 2 and 3 obtained 3 （1 insertion, 2 deletion）， 13 （8 insertion, 5 deletion） and 7 （1 insertion, 6 deletion） mutation types with the mutation efficiency being 25.71%, 94.28%, and 71.43%, respectively. Alizarin red bone staining and X-ray scan analysis showed that IBs were not existed in runx2b mutant F1 individuals, and micro-CT analysis found that the mineralization of other bone in runx2b mutant was not significantly affected compared to wild type fish （P > 0.05）。 RT-qPCR results also exhibited that bone-related genes had a lower expression level in runx2b mutants compared to wild type fish. Subsequently, we used the national standard methods to detect nutrition composition of muscle tissue in wild type （with IBs） and runx2b mutant F1 individuals （without IBs） at 120 dpf. The results showed that the basic muscle nutritional composition （moisture, crude protein, crude lipid and crude ash）， and the contents of most amino acids and fatty acids had no significant differences between wild and runx2b mutant at juvenile stage （P > 0.05）。 This study proves that the runx2b gene has an essential role for IBs formation in aquaculture species and lays an important foundation for obtaining a new blunt snout bream strain without IBs, which provides a good example for the application of IBs-free characters from model fish to commercial fish.

 

　　原文鏈接：https://doi.org/10.1016/j.aquaculture.2023.739263

 

　　Blunt snout bream （Megalobrama amblycephala） is an important herbivorous freshwater economic fish cultivated in China. Its economic value has been affected by the existence of intermuscular bones （IBs）。 Our previous study proved that runx2b is the key gene for the IBs formation, and lack of runx2b gene could obtain a stable zebrafish mutant without IBs. We found runx2b mutant F0 generation of blunt snout bream through CRISPR/Cas9 editing technology had a significant reduction on the number of IBs. In order to further obtain the blunt snout bream without IBs, we used the runx2b mutant F0 generation to self-bred to obtain F1 generation. Subsequently, the 35 runx2b mutant F1 individuals of blunt snout bream without IBs were genotyped and found the number of runx2b targeting sites 1, 2 and 3 obtained 3 （1 insertion, 2 deletion）， 13 （8 insertion, 5 deletion） and 7 （1 insertion, 6 deletion） mutation types with the mutation efficiency being 25.71%, 94.28%, and 71.43%, respectively. Alizarin red bone staining and X-ray scan analysis showed that IBs were not existed in runx2b mutant F1 individuals, and micro-CT analysis found that the mineralization of other bone in runx2b mutant was not significantly affected compared to wild type fish （P > 0.05）。 RT-qPCR results also exhibited that bone-related genes had a lower expression level in runx2b mutants compared to wild type fish. Subsequently, we used the national standard methods to detect nutrition composition of muscle tissue in wild type （with IBs） and runx2b mutant F1 individuals （without IBs） at 120 dpf. The results showed that the basic muscle nutritional composition （moisture, crude protein, crude lipid and crude ash）， and the contents of most amino acids and fatty acids had no significant differences between wild and runx2b mutant at juvenile stage （P > 0.05）。 This study proves that the runx2b gene has an essential role for IBs formation in aquaculture species and lays an important foundation for obtaining a new blunt snout bream strain without IBs, which provides a good example for the application of IBs-free characters from model fish to commercial fish.